The authors would like to thank Prof. Dr. Stefan Vieths of the Department of Molecular Allergology, Paul-Ehrlich-Institut, Langen, Germany, for providing the humanized/Fc&#949;RI-transfected RBL cells and for giving his consent to write this research methodology paper. We want to thank Prof. Dr. Fatima Ferreira for providing excellent feedback. We further would like to thank Prof. Dr. Ronald van Ree and Dr. Jaap Akkerdaas of the Department of Experimental Immunology, Amsterdam University Medical Centers, location AMC, Amsterdam, The Netherlands, for giving their consent to publish representative data provided in this methods paper, which were generated in the course of the project BM4SIT - innovations for allergy (www.BM4SIT.eu). The work of the authors has been supported by the Austrian Science Fund (Project P32189), by the University of Salzburg priority program Allergy-Cancer-BioNano Research Centre, by the doctoral program Immunity in Cancer and Allergy-ICA funded by the Austrian Science Fund (FWF W01213), and by the BM4SIT project (grant number 601763) from the European Union&#39;s Seventh Framework Program FP7.

Ethical approval to use sera derived from birch pollen allergic patients was obtained from the Dutch ethical committee (approval number: NL65758.018.18).
1. Safety procedures
<ol>
	<li>Work under sterile conditions using a biological safety class 2 workbench during the first day of the experiment (Biosafety Level 2). Follow the safety guidelines of the institution for the usage of human serum.</li>
</ol>
2. Complement inactivation of human sera
<ol>
	<li>Harvest a dense culture of P3X63Ag8.653 cells (termed Ag8 cells henceforth), from the cell culture flask and transfer them into a centrifugation tube.
	<ol>
		<li>Use the following culture medium for these cells: Modified Eagles&#180;s Minimum Essential Medium with reduced serum concentration, 1% Penicillin-Streptomycin (100 units Pen., 0.1 mg/mL Strep.), 5% heat-inactivated fetal calf/bovine serum (FCSi).</li>
	</ol>
	</li>
	<li>Centrifuge Ag8 cells for 5 min at 250 x g at room temperature.</li>
	<li>Re-suspend the cell pellet to a final concentration of approximately 1 x 106 cells/mL in huRBL medium (Minimum Essential Medium Eagle with Alpha Modification, 4 mM L-Glutamine, 5% FCSi, 1% G418 (100% stock: 10 g/125 mL dH2O). 
	NOTE: Maintain Ag8 cells by passaging for future use as well.</li>
	<li>Dilute human sera 1:10 in Ag8 cell suspension. Final serum dilution in the assay will be 1:20. 
	NOTE: For sera with low specific IgE a 1:5 (1:10 final dilution) can be used.</li>
	<li>Incubate for 1 h at 37 &#176;C and 5%-7% CO2.</li>
</ol>
3. Harvesting and seeding of huRBL cells
<ol>
	<li>Aspirate the medium from a T-75 cell culture flask carefully without touching the huRBL cells (huRBL cells are adherent). Ensure that the cells are around 50%-90% confluent. 
	NOTE: Depending on the cell confluence, the cell content of a dense T-75 cell culture flask is usually enough for one to two 96-well plates.</li>
	<li>Wash the cells twice by adding 10 mL of Dulbecco&#39;s phosphate-buffered saline (DPBS). Add DPBS to the opposite side of the flask and not directly onto the cells.</li>
	<li>Aspirate DPBS and add 5 mL of pre-warmed 1x trypsin-EDTA (0.05%/0.02% EDTA diluted in DPBS) for cell detachment.</li>
	<li>Incubate the flask for 5 min at 37 &#176;C.</li>
	<li>Detach cells by carefully tapping the flask.</li>
	<li>Transfer the cell suspension into a 15 mL centrifugation tube and fill up with huRBL medium or DPBS to dilute the trypsin-EDTA.</li>
	<li>Centrifuge the cells at 250 x g for 5 min at room temperature.</li>
	<li>Aspirate the supernatant and resuspend the pellet in 5 mL of huRBL medium for cell counting.</li>
	<li>Count the cells and dilute them in huRBL medium to obtain a final concentration of 2 x 106 cells/mL.</li>
	<li>Use a sterile 96-well plate and add 50 &#181;L of huRBL cell suspension per well, which is equivalent to 1 x 105 cells/well.</li>
</ol>
4. Passive sensitization of huRBL cells
<ol>
	<li>Centrifuge the pre-incubated Ag8/serum suspension for 5 min at 250 x g.</li>
	<li>Transfer 50 &#181;L of the centrifuged Ag8/serum suspension to each well containing huRBL cells without disturbing the Ag8 cell pellet.
	<ol>
		<li>Include the no antigen control, which are sensitized, but unstimulated cells (do not add antigen), serving as an indication for the bottom signal plateau/background. Background and maximum lysis control wells do not need to be sensitized with serum. Add 50 &#181;L of huRBL medium to control wells instead.</li>
	</ol>
	</li>
	<li>Cover the plate with the lid and incubate overnight at 37 &#176;C and 5%-7% CO2.</li>
</ol>
5. Antigen-stimulated degranulation and mediator release
<ol>
	<li>Prepare the antigen dilution in 1x Tyrode&#39;s buffer (9.5 g/L Tyrode&#39;s salts, 0.1% bovine serum albumin (BSA), 0.5 g/L Sodium hydrogen carbonate (NaHCO3) in dH2O) in advance. A final amount of 100 &#181;L per well is needed. 
	NOTE: Not every allergen, either purified from natural sources or recombinantly produced, might be stable in 1x Tyrode&#39;s buffer. Therefore, perform stability tests in 1x Tyrode&#39;s buffer prior to the assay procedure. Alternatively, dilute 1x Tyrode&#39;s buffer in deuterium oxide (D2O) to increase the signal-to-noise ratio of the assay.</li>
	<li>Make 8 dilutions of the antigen of interest of a 1:10 dilution series in reaction tubes starting with either 10 or 1 &#181;g/mL of protein. 
	NOTE: Always test the dilution series beforehand. Alternatively, adapt the 1:10 dilution series (e.g., 1:5, 1:20, or 1:30) in order to cover the full release curve. In addition, the starting concentration can vary depending on the experimental setup.</li>
	<li>To wash huRBL cells plated on the 96-well plate, remove the sera-containing cell medium first by carefully aspirating, inverting, and tapping the plate on absorbent paper.</li>
	<li>Wash cells with 200 &#181;L of 1x Tyrodes&#39;s buffer per well. Treat all wells similarly. 
	NOTE: Add the washing solution slowly to the cells in order to not disturb them.</li>
	<li>Leave it for approximately 30 s and repeat the washing step three times in total.</li>
	<li>After adding the washing solution for the final time, leave the solution in the wells until ready to continue with adding the antigen dilution. 
	NOTE: Avoid exposing cells to air for too long.</li>
	<li>Transfer 100 &#181;L of antigen solution to each well containing the pre-sensitized huRBL cells. 
	NOTE: If analyzing several different parameters, transfer the individual samples of the dilution series into an additional non-binding 96-well plate (use the same layout as on the huRBL plate) and transfer them afterward with a multichannel pipette directly on the huRBL cell plate. This way, exposing the cells to air for too long can be avoided, which might result in poor assay performance (lower/no signal).</li>
	<li>Cover control wells (maximum lysis and non-sensitized background cells) with 100 &#181;L of 1x Tyrode&#39;s buffer. Do not stimulate these control wells with the antigen.
	<ol>
		<li>Additionally, add 100 &#181;L of 1x Tyrode&#39;s buffer to the sensitized no-antigen wells of the dilution series, which is needed to take antigen-independent spontaneous release of sensitized cells into account during data analysis.</li>
	</ol>
	</li>
	<li>Incubate huRBL cells for 1 h at 37 &#176;C and 5%-7% CO2.</li>
</ol>
6. Fluorescence measurement of &#946;-hexosaminidase activity
<ol>
	<li>Treat the wells of the maximum lysis control with 10 &#181;L of 10% Triton X-100 per well and mix properly in order to lyse the cells completely and obtain the 100% release of &#946;-hexosaminidase.</li>
	<li>Add 50 &#181;L of substrate solution into a new non-binding 96-well plate. Substrate solution for one 96-well plate: 5 mL of 0.1 M citric assay buffer, pH 4.5; and 80 &#181;L of 10 mM 4-methylumbelliferyl N-acetyl-&#946;-D-glucosaminide.</li>
	<li>Transfer 50 &#181;L of cell supernatant of all wells to the new plate containing the substrate solution. 
	NOTE: Pipette the supernatant carefully off the huRBL plate in order to not disrupt the huRBL cells.</li>
	<li>Incubate the plate with substrate solution and cell supernatant for 1 h at 37 &#176;C to allow conversion of the fluorogenic substrate. 
	NOTE: Keep the huRBL plate for cell viability assay.</li>
	<li>Add 100 &#181;L of stopping solution (15 g/L glycine, 11.7 g/L NaCl dissolved in dH2O, pH 10.7) per well.</li>
	<li>Measure the fluorescence at 360 nm excitation and 465 nm emission using a plate reader.</li>
</ol>
7. Data analysis
<ol>
	<li>For basic calculations of percentage release, use any spreadsheet software.</li>
	<li>For the background subtraction/baseline removal, subtract the average of the background wells from all other wells.</li>
	<li>Calculate the mean of maximum lysis wells and express data of the dilution series in percentage. This way one can express data as a percentage of cell release normalized to the maximum enzyme release caused by cell lysis.</li>
	<li>Complete dose-response mediator release curves are represented best as XY graphs with the antigen concentration on a log on the X-axis and percentage of mediator release on the Y-axis.</li>
	<li>Add the values of the no-antigen control as a dashed line to indicate the background or the bottom plateau. 
	NOTE: Several similarly treated sera can be compared using this normalization strategy. For direct comparison, it is further recommended to calculate the half maximum release, which is the antigen concentration (in ng/mL) necessary for half maximal release defined as the average of the maximal and minimal values per curve. The antigen concentration to stimulate half maximal release is calculated by interpolation of the half maximal release value into a logarithmic regression line.</li>
</ol>

<ol>
	<li>Curin, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next-generation+of+allergen-specific+immunotherapies:+molecular+approaches.">Next-generation of allergen-specific immunotherapies: molecular approaches.</a> Current Allergy and Asthma Reports. 18 (7), 39 (2018).</li><li>Okamoto-Uchida, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+results+of+IgE+binding-+and+crosslinking-Based+allergy+tests+caused+by+allergen+immobilization.">Different results of IgE binding- and crosslinking-Based allergy tests caused by allergen immobilization.</a> Biological and Pharmaceutical Bulletin. 39 (10), 1662-1666 (2016).</li><li>Vogel, L., L&#252;ttkopf, D., Hatahet, L., Haustein, D., Vieths, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+functional+in+vitro+assay+as+a+novel+tool+for+the+standardization+of+allergen+extracts+in+the+human+system.">Development of a functional in vitro assay as a novel tool for the standardization of allergen extracts in the human system.</a> Allergy. 60 (8), 1021-1028 (2005).</li><li>Ocmant, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basophil+activation+tests+for+the+diagnosis+of+food+allergy+in+children.">Basophil activation tests for the diagnosis of food allergy in children.</a> Clinical and Experimental Allergy. 39 (8), 1234-1245 (2009).</li><li>Platts-Mills, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+immunoglobulin+E+in+allergy+and+asthma.">The role of immunoglobulin E in allergy and asthma.</a> American Journal of Respiratory and Critical Care Medicine. 164 (8), 1-5 (2001).</li><li>Galli, S. J., Tsai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IgE+and+mast+cells+in+allergic+disease.">IgE and mast cells in allergic disease.</a> Nature Medicine. 18 (5), 693-704 (2012).</li><li>Lawrence, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Half-life+of+IgE+in+serum+and+skin:+Consequences+for+anti-IgE+therapy+in+patients+with+allergic+disease.">Half-life of IgE in serum and skin: Consequences for anti-IgE therapy in patients with allergic disease.</a> The Journal of Allergy and Clinical Immunology. 139 (2), 422-428 (2017).</li><li>Draber, P., Halova, I., Polakovicova, I., Kawakami, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+transduction+and+chemotaxis+in+mast+cells.">Signal transduction and chemotaxis in mast cells.</a> European Journal of Pharmacology. 778, 11-23 (2016).</li><li>Peebles, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prostaglandins+in+asthma+and+allergic+diseases.">Prostaglandins in asthma and allergic diseases.</a> Pharmacology &amp; Therapeutics. 193, 1-19 (2019).</li><li>Cyphert, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allergic+inflammation+induces+a+persistent+mechanistic+switch+in+thromboxane-mediated+airway+constriction+in+the+mouse.">Allergic inflammation induces a persistent mechanistic switch in thromboxane-mediated airway constriction in the mouse.</a> American Journal of Physiology. Lung Cellular and Molecular Physiology. 302 (1), 140-151 (2012).</li><li>M&#233;ndez-Enr&#237;quez, E., Hallgren, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mast+cells+and+their+progenitors+in+allergic+asthma.">Mast cells and their progenitors in allergic asthma.</a> Frontiers Immunology. 10, 821 (2019).</li><li>Demoly, P., Lebel, B., Arnoux, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allergen-induced+mediator+release+tests.">Allergen-induced mediator release tests.</a> Allergy. 58 (7), 553-558 (2003).</li><li>Yamaga, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreased+intracellular+histamine+concentration+and+basophil+activation+in+anaphylaxis.">Decreased intracellular histamine concentration and basophil activation in anaphylaxis.</a> Allergology International. 69 (1), 78-83 (2020).</li><li>Huang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+assay+based+on+particle+analysis+for+cell+degranulation+detection+in+basophils+and+mast+cells.">A rapid and sensitive assay based on particle analysis for cell degranulation detection in basophils and mast cells.</a> Pharmacological Research. 111, 374-383 (2016).</li><li>Gonz&#225;lez-P&#233;rez, R., Poza-Guedes, P., Barrios Del Pino, Y., Matheu, V., S&#225;nchez-Mach&#237;n, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+major+mite+allergens+from+European+standardized+commercial+extracts+for+in+vivo+diagnosis:+addressing+the+need+for+precision+medicine.">Evaluation of major mite allergens from European standardized commercial extracts for in vivo diagnosis: addressing the need for precision medicine.</a> Clinical and Translational Allergy. 9, 14 (2019).</li><li>Focke, M., Marth, K., Valenta, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+composition+and+biological+activity+of+commercial+birch+pollen+allergen+extracts.">Molecular composition and biological activity of commercial birch pollen allergen extracts.</a> European Journal of Clinical Investigation. 39 (5), 429-436 (2009).</li><li>Hoffmann, A., Vieths, S., Haustein, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biologic+allergen+assay+for+in+vivo+test+allergens+with+an+in+vitro+model+of+the+murine+type+I+reaction.">Biologic allergen assay for in vivo test allergens with an in vitro model of the murine type I reaction.</a> The Journal of Allergy and Clinical Immunology. 99 (2), 227-232 (1997).</li><li>Sun, N., Zhou, C., Zhou, X., Sun, L., Che, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+rat+basophil+leukemia+(RBL)+cell-based+immunological+assay+for+allergen+identification,+clinical+diagnosis+of+allergy,+and+identification+of+anti-allergy+agents+for+use+in+immunotherapy.">Use of a rat basophil leukemia (RBL) cell-based immunological assay for allergen identification, clinical diagnosis of allergy, and identification of anti-allergy agents for use in immunotherapy.</a> Journal of Immunotoxicology. 12 (2), 199-205 (2015).</li><li>Kaul, S., Hoffmann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mediator+release+assay+of+rat+basophil+leukemia+cells+as+alternative+for+passive+cutaneous+anaphylaxis+testing+(PCA)+in+laboratory+animals.">Mediator release assay of rat basophil leukemia cells as alternative for passive cutaneous anaphylaxis testing (PCA) in laboratory animals.</a> Altex. 18 (1), 55-58 (2001).</li><li>Passante, E., Frankish, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RBL-2H3+cell+line:+its+provenance+and+suitability+as+a+model+for+the+mast+cell.">The RBL-2H3 cell line: its provenance and suitability as a model for the mast cell.</a> Inflammation Research. 58 (11), 737-745 (2009).</li><li>Kaul, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mediator+release+assays+based+on+human+or+murine+immunoglobulin+E+in+allergen+standardization.">Mediator release assays based on human or murine immunoglobulin E in allergen standardization.</a> Clinical and Experimental Allergy. 37 (1), 141-150 (2007).</li><li>Huang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+in+vitro+and+in+vivo+models+in+the+study+of+food+allergy.">Application of in vitro and in vivo models in the study of food allergy.</a> Food Science and Human Wellness. 7 (4), 235-243 (2018).</li><li>Klueber, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homologous+tropomyosins+from+vertebrate+and+invertebrate:+Recombinant+calibrator+proteins+in+functional+biological+assays+for+tropomyosin+allergenicity+assessment+of+novel+animal+foods.">Homologous tropomyosins from vertebrate and invertebrate: Recombinant calibrator proteins in functional biological assays for tropomyosin allergenicity assessment of novel animal foods.</a> Clinical and Experimental Allergy. 50 (1), 105-116 (2020).</li><li>Zhang, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+thermal+processing+effects+on+peanut+allergenicity.">Different thermal processing effects on peanut allergenicity.</a> Journal of the Science of Food and Agriculture. 99 (5), 2321-2328 (2019).</li><li>Soh, W. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+roles+of+Bet+v+1+ligands+in+allergen+stabilization+and+modulation+of+endosomal+protease+activity.">Multiple roles of Bet v 1 ligands in allergen stabilization and modulation of endosomal protease activity.</a> Allergy. 74 (12), 2382-2393 (2019).</li><li>Wolf, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amb+a+1+isoforms:+Unequal+siblings+with+distinct+immunological+features.">Amb a 1 isoforms: Unequal siblings with distinct immunological features.</a> Allergy. 72 (12), 1874-1882 (2017).</li><li>Eichhorn, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+design,+structure-activity+relationship,+and+immunogenicity+of+hypoallergenic+Pru+p+3+variants.">Rational design, structure-activity relationship, and immunogenicity of hypoallergenic Pru p 3 variants.</a> Molecular Nutrition &amp; Food Research. 63 (18), 1900336 (2019).</li><li>Abd Rani, N. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanistic+studies+of+the+antiallergic+activity+of+phyllanthus+amarus+Schum.+and+Thonn.+and+its+compounds.">Mechanistic studies of the antiallergic activity of phyllanthus amarus Schum. and Thonn. and its compounds.</a> Molecules. 26 (3), (2021).</li><li>Huber, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+clinical+outcome+of+birch+pollen+immunotherapy+relate+to+induction+of+blocking+antibodies+preventing+IgE+from+allergen+binding?+A+pilot+study+monitoring+responses+during+first+year+of+AIT.">Does clinical outcome of birch pollen immunotherapy relate to induction of blocking antibodies preventing IgE from allergen binding? A pilot study monitoring responses during first year of AIT.</a> Clinical and Translational Allergy. 8 (1), 39 (2018).</li><li>Araujo, G. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF&#946;1+mimetic+peptide+modulates+immune+response+to+grass+pollen+allergens+in+mice.">TGF&#946;1 mimetic peptide modulates immune response to grass pollen allergens in mice.</a> Allergy. 75 (4), 882-891 (2020).</li><li>Passante, E., Frankish, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deficiencies+in+elements+involved+in+TLR4-receptor+signalling+in+RBL-2H3+cells.">Deficiencies in elements involved in TLR4-receptor signalling in RBL-2H3 cells.</a> Inflammation Research. 59, 185-186 (2010).</li></ol>

The authors have nothing to disclose.

The herein described huRBL cell-based mediator release assay is a robust method that can easily be performed and implemented in any laboratory. The only requirement is that cells need to be cultivated under sterile conditions. The assay is used to assess the likelihood of an allergen or allergenic source to evoke patients&#39; IgE-crosslinking and basophil degranulation17. The assay can easily be adapted to any allergen or allergenic source as long as the patient&#180;s serum with a high level of specific IgE, recognizing the allergen of interest, is available. It is recommended to perform a cell viability assay in addition to the mediator release assay in order to account for any potential cytotoxic effects that might result in poor assay performance. This might be due to incomplete complement-inactivation of the sera or other serum-derived cytotoxic effects. Even the antigen itself, for instance, because of proteolytic/enzymatic activity, can harm the huRBL cells. We are usually using a cell viability assay with MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) to evaluate potential cytotoxic effects. The assay can easily be performed with the huRBL cells left after the cell supernatant was collected and transferred (see step 6.3. of the protocol). Compared to other immunochemical methods, such as ELISAs and western blotting, for determining the allergenic potential of either individual allergens or complex extracts based on allergen-IgE binding, this assay can detect not only the binding of IgE to an allergen but can also measure the functionality of both, human IgE and the allergen, to provoke IgE-mediated basophil degranulation18. Thus, it can aid in studying the severity of allergic symptoms ex vivo using patients&#39; sera. The assay is reported to be more consistent and efficient than the classical passive cutaneous anaphylaxis tests since the assay utilizes the RBL-2H3 cells, which are relatively easy to handle and produce less variability in results compared to primary cells, such as mast cells or human basophils19,20. In addition to this, the assay provides a good representation of the biological activity of allergens and can accurately estimate the total allergen content in a given complex sample3. For troubleshooting certain steps in the protocol please refer to Table 1.
Regarding the applicability of this version of the mediator release assay, it has mostly been used for research purposes but also for the standardization of allergenic extracts based on their biological activity. This includes the analysis of different batches of SPT solutions, provocation test solution, as well as extracts used for allergen-specific immunotherapy; as shown for pollen, cat dander, house dust mite, and peanut extracts, as well as bee venom3,17,21. The technique can be applied especially in diagnosing food allergies, as it can detect even minimal amounts of allergenic constituents in complex food products such as peanuts, milk, wheat, and eggs22. In this respect, is has also been reported as a valuable tool for the assessment of allergenicity of animal food allergens, such as tropomyosins, and can aid in distinguishing potent allergens from non-allergens23. As a research tool, the assay is used to study the impact of food processing as well as to evaluate the influence of ligand binding to allergens and its effect on allergenicity24,25. For instance, the binding of Bet v 1 to ligands was shown not to affect the allergen-IgE cross-linking, although it caused an increase in its thermal and proteolytic stability25. The assay can be used to compare patient&#39;s reactivity to minor and major allergens, as well as to investigate the cross-reactivity of allergen homologues and isoforms, as shown in our example using Bet v 1 and the homologous food allergen Cor a 1 (Figure 3). Regarding allergen isoforms, the mediator release assay was used to identify the major allergen Amb a 1.01 as the most potent IgE-reactive isoform in ragweed pollen (Ambrosia artemisiifolia). In comparison, the other two identified isoforms in ragweed pollen extracts, Amb a 1.02 and Amb a 1.03, showed reduced reactivity to patients&#39; IgE26.
In recent years, the assay has been applied to study potential anti-allergic compounds and novel hypoallergenic variants of allergens, aiding in the identification of suitable candidates for allergen-specific immunotherapy27,28. Another novel approach is to use the assay to monitor treatment efficacy in the course of allergen-specific immunotherapy. In this regard, our research group developed a huRBL assay inhibition system, which correlated well with the reduction of the patient&#39;s symptom score during allergen-specific immunotherapy29. The assay has also been proposed to study the immunosuppressive effects of TGF&#946;1 on allergen-induced IgE-mediated degranulation30.
The limitations of the assay are that even though the huRBL cells possess some features of mast cells or basophils, they do not completely mimic the natural function of these effector cells. For example, mast cells widely express the pattern recognition receptor Toll-like receptor 4 (TLR4), necessary for pathogen recognition, whereas it is completely deficient in the RBL-2H3 cells31. Due to this difference in functionality, the assay does not fully mimic the real-life situation, which needs to be kept in mind when interpreting the data. In addition, since the huRBL cells are cancerous basophilic cells, changes in culture conditions and prolonged culturing can lead to phenotypic differences leading to altered results among different laboratories20. Another aspect is the choice of allergen concentration that has to be taken into account when adapting this method since high allergen concentrations might result in non-IgE mediated degranulation due to the presence of high amounts of proteases or endotoxins18. Other limitations are the dependency on human sera with relatively high specific IgE levels (RAST class 5-6), and the need for cell culture systems, which remains an obstacle that needs to be overcome in order to implement the technique in the daily clinical routine.
Apart from these limitations, the huRBL assay represents a valuable research tool for the diagnosis and treatment of allergic diseases and can be used in a wide range of applications.

Type I hypersensitivity reactions, characterized by Immunoglobulin E (IgE) production specific for a respective antigen, affect nearly a third of the world&#39;s population. These reactions are associated with several allergic manifestations such as asthma and rhinoconjunctivitis and can even lead to systemic life-threatening reactions1. Unlike in vivo tests, immunochemical approaches, such as the enzyme-linked immunosorbent assay (ELISA), are solely suitable for investigating the target binding of antibodies but do not address the functional aspect of proteins that can cause immediate hypersensitivity reactions. The immobilization of the allergens on solid supports (e.g., ELISA plates) could cause changes in their structural integrity and the destruction of allergy relevant epitopes2. Even skin prick tests (SPT), the most common tool to confirm sensitization against certain allergens, have their limits concerning the detection of symptomatic IgE-mediated food allergy or allergen availability3,4. In order to find an ethical, highly specific, sensitive, and cost-effective method for testing the biological potency of allergens to cause a type I hypersensitivity reaction, the so-called mediator release assays have been established.
The principle of these assays relies on events following the sensitization phase and the accompanying ability of IgE to bind to the &#945;-chain of the high-affinity receptors expressed on the surface of effector cells, such as mast cells and basophils. IgE is mainly produced by plasma cells in the mucosal-associated lymphoid tissue. Although it is the least abundant immunoglobulin (around 0.05% in non-atopic individuals) in the blood, it possesses an extraordinarily high biological activity being the main cause for allergic symptoms. The half-life of IgE can increase from 2-3 days to several weeks and even months when bound to its receptors on effector cells. Subsequent binding of an antigen to the variable region of two receptor-bound IgE molecules leads to their cross-linking followed by the induction of a downstream signaling cascade in the effector cell leading to degranulation and the release of several pro-inflammatory mediators causing vasodilation, such as histamine, serine proteases (e.g., tryptase), and prostaglandins5,6,7. The secretion of cytokines such as interleukin 4 (IL-4) and IL-13 are responsible for the maintenance of the inflammatory T helper 2 (Th2) response and the class-switching of B cells to IgE-producing plasma cells5,8,9. On the other hand, released thromboxane causes bronchoconstriction, and leukotrienes stimulate smooth muscle contraction as well as vascular leakage, and play a crucial role in airway inflammation leading to asthma or allergic rhinitis10,11.
Research tools for analyzing most of the aforementioned mediators have been established, although with some major disadvantages. Tryptase assays are suitable clinical approaches for the measurement of systemic anaphylaxis through mast cell activation but their sensitivity and specificity in allergy diagnoses is too inaccurate compared to gold standard methods such as SPT. On the other hand, cysteinyl leukotriene assays are not capable of diagnosing allergies to &#946;-lactams or nonsteroidal anti-inflammatory drugs12. Protocols for the measurement of histamine as a major mediator released in allergic reactions were already established in the 1960s. Once released in the peripheral blood, histamine is immediately degraded by histamine methyltransferases resulting in a plasma half-life of only a few minutes, making its analysis quite challenging13. Aside from its instability, the monitoring of histamine was shown to have a low specificity and sensitivity for drug allergies as well as commercial food proteins and wasp venoms12.
In vitro models with effector cell lines have been introduced as an alternative to the labor-intensive procedures of isolation and cultivation of basophils from allergic patients to perform release assays. Therefore, the rat basophilic leukemia- (RBL-) based assay using the RBL-2H3 cell line has been established3. Since this cell line is not capable of binding human IgE, it was first transfected with the &#945;-, &#946;-, and &#947;-chain of the human IgE plasma-membrane receptor (Fc&#949;RI). Several clones have been generated and tested for expression levels and homogeneity of the human &#945;-chain, of which the clone RBL-30/25 emerged as the most promising candidate for in vitro testing. The signaling cascade induced upon receptor activation of the transfected clone was tested via calcium mobilization assays. As an indicator for degranulation and surrogate for histamine release, the lysosomal enzyme &#946;-hexosaminidase was measured, which has the significant advantage of higher stability14. The mediator release using RBL-30/25 cells reaches up to 100% and is, therefore, used to test sera derived from allergic patients. The assay was tested for the mediator release after challenging sensitized cells with commercial allergen extracts. This led to the finding that there is a tremendous variation in the composition (of up to 60-fold regarding the total protein content) of allergen extracts derived from different manufacturers and used for diagnostic (e.g., SPT) or therapeutic approaches3,15,16.
Herein, we provide a detailed description of the RBL protocol to perform the mediator release assay using serum from allergic donors. During passive sensitization, IgE in the serum is captured by the high affinity Fc&#949;R1 receptor expressed on the surface of the basophilic cells. Upon antigen-stimulation, bound IgEs specific for the antigen are cross-linked, triggering cell degranulation and the release of the mediator &#946;-hexosaminidase. The activity of &#946;-hexosaminidase is subsequently measured using a suitable substrate. For the assay, huRBL-2H3 cells were used, and termed huRBL in the following protocol. The protocol describes a standard antigen dilution series with 8 steps diluted 1:10 ranging from 1 &#181;g/mL to 0.1 pg/mL of allergen.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4-Methylumbelliferyl N-acetyl-&#946;-D-glucosaminide</td>
			<td>Sigma</td>
			<td>M2133</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate for huRBL cells (Nunc MicroWell 96-Well, Nunclon Delta-treated, flat-bottom microplate)</td>
			<td>ThermoFisher Scientific</td>
			<td>167008</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate for substrate solution and cell supernatant (Greiner Bio-One non-treated 96-well microplates)</td>
			<td>Fisher Scientific</td>
			<td>655101</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>10735078001</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric acid</td>
			<td>Applichem</td>
			<td>131018</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate-buffered saline (DPBS without calcium and magnesium)</td>
			<td>Sigma</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>G418</td>
			<td>Sigma</td>
			<td>A1720</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Applichem</td>
			<td>A3707</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat-inactivated fetal calf/bovine serum (FCSi)</td>
			<td>Sigma</td>
			<td>F0804</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine (200 mM)</td>
			<td>Sigma</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimum Essential Medium Eagle with Alpha Modification, with ribonucleosides, deoxyribonucleosides and sodium bicarbonate, without L-glutamine, liquid, sterile-filtered, suitable for cell culture</td>
			<td>Sigma</td>
			<td>M8042</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM reduced serum medium, GlutaMAX supplement</td>
			<td>Gibco/ThermoFisher Scientific</td>
			<td>51985034</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10K units Pen.&#160; 10 mg/mL Strep.)</td>
			<td>Sigma</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Applichem</td>
			<td>A2942</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydrogen carbonate (NaHCO3)</td>
			<td>Applichem</td>
			<td>131638</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Sigma</td>
			<td>59418C</td>
			<td></td>
		</tr>
		<tr>
			<td>Tyrode&#8217;s salt</td>
			<td>Sigma</td>
			<td>T2145</td>
			<td></td>
		</tr>
	</tbody>
</table>

humanized mediator release assay as a read-out for allergen potency

Mediator release assays analyze in vitro immunoglobulin E (IgE)-mediated degranulation and secretion of mediators by effector cells, such as mast cells and basophils, upon stimulation with serial dilutions of putative allergens. Therefore, these assays represent an essential tool that mimics the in vivo degranulation process, which occurs upon allergen exposure in sensitized patients or in skin prick tests. Additionally, these assays are usually employed to investigate the allergenic potential of proteins and the reactivity of patients' sera's reactivity. Herein, we describe a simple 2-day protocol using an immortalized rat basophil leukemia cell line transfected and humanized with the human high-affinity IgE plasma-membrane receptor (Fc&#949;RI). This variant of the mediator release assay is a robust, sensitive, and reproducible in vitro cell-based system without the need to immobilize the antigen to solid matrices. The protocol consists of the following steps: (1) complement inactivation of human sera, (2) harvesting, seeding, and passive sensitization of the cells, (3) stimulation with antigen to cause mediator release, and (4) measuring of &#946;-hexosaminidase activity as a surrogate for the released inflammatory mediators, such as histamine. The assay represents a useful tool to assess the capacity of the allergen-IgE cross-linking to trigger cell degranulation and can be implemented to standardize allergen extracts, to compare patients' reactivity to minor or major allergens and to allergenic extracts (pollen, cat dander, etc.), to investigate the potency of allergen homologs, isoforms, and fold-variants (e.g., hypoallergenicity), as well as the effects of ligands on the allergenic activity. A more recent application includes the use of the assay to monitor the treatment efficacy in the course of allergen immunotherapy.

The mediator release assay, based on huRBL cells (Figure 1A and B), results in a bell-shaped dose-response curve (Figure 1C). For simplified data representation, the antigen concentration necessary for the half maximum mediator release can be calculated using linear regression (Figure 1D). A cell viability assay is performed to exclude cytotoxic effects derived from either the sensitizing serum or the antigen used for stimulation (Figure 1E). The assay can be used to test the reactivity of different sera to a certain antigen. In our case, 4 out of 5 sera, derived from birch pollen allergic patients, responded to Bet v 1 stimulation. Serum #1 showed the highest mediator release (Figure 2). Serum #5 did not respond to Bet v 1 stimulation and, thus, might react to other birch pollen allergens (e.g., Bet v 2, profilin). These data indicate that Bet v 1 is a potent allergen responsible for IgE-mediated allergic symptoms. By using the huRBL assay, the cross-reactivity of IgE to homologous allergens can be assessed (Figure 3). Here, both birch pollen allergic patients responded well to Bet v 1, whereas only patient #2 responded also to Cor a 1, the Bet v 1-homologous food allergen found in hazelnuts. Based on these data, patient #2 most likely has higher Cor a 1-cross-reactive IgE levels than patient #1, resulting in oral allergy symptoms upon hazelnut consumption. Even the assessment of the hypoallergenic nature of mutant variants of allergens (decreased potency) can be analyzed and compared to their wild-type counterpart (Figure 4). In the provided example, the release curve of the fold variant shifted towards a higher antigen concentration compared to the wild-type allergen, resulting in a significantly higher concentration of antigen necessary to provoke half maximal release (Figure 4B). These data imply that the generated mutant/fold variant is less allergenic compared to the wild-type protein. This reduced potency to trigger IgE-mediated degranulation highlights the hypoallergenic character of the fold variant. Based on this assay, the fold variant is an interesting candidate for allergen-specific immunotherapy since it might cause reduced IgE-associated side effects during the treatment.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62702/62702fig01.jpg" /> 
Figure 1: Humanized RBL cells and a representative bell-shaped curve of IgE-allergen cross-linking-induced &#946;-hexosaminidase release. RBL cells are adherent to the culture flasks, which gives them a rod-like shape as they are trying to attach themselves (A). An ideal level of confluence for cells to be harvested is no more than 90% (B). Cells are shown under magnification of 40x and 10x, respectively. Cells that were sensitized with human serum of a birch pollen allergic individual reacting upon challenge with recombinant Bet v 1 (rBet v 1), the major birch pollen allergen (C). As surrogate for mediator release, the &#946;-hexosaminidase activity is measured in cell supernatants. The bell-shaped curve results from a monovalent occupation of antigen epitopes on IgE due to the excess of allergen, which competitively inhibits the allergen-IgE cross-linking at high antigen concentrations. Another explanation for the low release at high allergen concentrations is the inhibition of intracellular pathways in presence of excess antigen. For determination of the allergen concentration necessary to obtain half maximal release, a logarithmic regression line based on the experimental values representing the linear part of the slope of the mediator release curve was used (D). The red dotted line represents the logarithmic regression line used for calculation. The formula of the regression line is shown in red. The half maximal release is defined as: half maximal release = (minimum release value + maximum release value)/2. In the example, the calculated half maximal release was 20.6%. The representative human serum used in this experiment was diluted 1:20 for incubation with huRBL cells, and the antigen concentration used for stimulation ranged from 100 &#181;g/mL to 0.004 pg/mL of Bet v 1. A cell viability assay, in this case a MTT assay, was performed with the remaining cells after antigen stimulation to assess the influence of the sensitizing serum as well as of the antigen dilution on cell viability and cell count (E). Untreated background cells and lysed cells (maximum lysis) are shown as dotted line. <a href="https://www.jove.com/files/ftp_upload/62702/62702fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62702/62702fig02.jpg" /> 
Figure 2: Representative curves of &#946;-hexosaminidase percent release of five different human sera. The same antigen concentration range of rBet v 1 was incubated with huRBL cells that were sensitized with sera of different birch pollen sensitized individuals. There is a clear difference of percent release between the different patients corresponding to the severity of their symptoms. Notice that patient #5 is non-reactive to the major birch pollen allergen Bet v 1. All five human sera used to obtain these mediator release curves were diluted equally 1:20 for incubation with huRBL cells. <a href="https://www.jove.com/files/ftp_upload/62702/62702fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62702/62702fig03.jpg" /> 
Figure 3: Cross-reactivity of IgE derived from sera of birch pollen sensitized patients with the Bet v 1 homologous hazelnut allergen Cor a 1. Two representative sera of patients sensitized to birch pollen strongly react to Bet v 1 as well as to a lesser degree to the homologous allergen Cor a 1. Patient 2 is shows a significant reaction to Cor a 1, and thus will likely exhibit oral allergy symptoms upon hazelnut consumption, compared to patient 1 where the mediator release is almost negligible. The dotted line represents the no-antigen control, which are cells sensitized with the human sera but not stimulated with an allergen, and, thus, serves as indication for the bottom signal plateau/background. <a href="https://www.jove.com/files/ftp_upload/62702/62702fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62702/62702fig04.jpg" /> 
Figure 4: Comparison of percent release between rBet v 1 wild type and a hypoallergenic fold-variant. The same serum of a birch pollen-sensitized individual was incubated with rBet v 1 wild type (wt) and a hypoallergenic fold-variant of the major birch pollen allergen (A). Even though mediator release is seen in both antigens, there is a clear shift toward higher antigen concentrations when comparing the fold-variant to wild type rBet v 1 for the same percent release. A standard way of comparing the difference in percent release of different antigens is calculating the concentration of antigen needed to attain half maximum release (B). This is usually performed in biological replicates (testing of the same antigen range for each allergen in different human sera). Usually, in order to draw any significant conclusions, the mediator release is performed with sera from at least 8 to 10 different patients. Here the results of four different sera are plotted as an example. A paired t-test was used for statistical analysis. *p &#8804; 0.05; **p &#8804; 0.01; ***p &#8804; 0.001; ****p &#8804; 0.0001. <a href="https://www.jove.com/files/ftp_upload/62702/62702fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1" fo:keep-with-next.within-page="always" fo:text-align="left">
	<tbody>
		<tr>
			<td>Potential questions and troubleshooting</td>
			<td>Solution</td>
		</tr>
		<tr>
			<td rowspan="2">Assay-to-assay variability due to altered cell responsiveness</td>
			<td>Ensure that the cell passage cycle number does not exceed 20 to 30 passages. Make frozen stocks at early passages for future experiments.&#160;</td>
		</tr>
		<tr>
			<td>Rather rely on biological replicates (use of different sera) than technical ones.</td>
		</tr>
		<tr>
			<td>Sera contains low levels of specific IgE</td>
			<td>A lower final serum dilution can be used (i. e. 1:10) instead of 1:20. Conversely, sera containing high levels of specific IgE can be further diluted (1:30 or 1:40).</td>
		</tr>
		<tr>
			<td>Not enough cells to perform the assay</td>
			<td>Make sure the confluency in a T-75 flask is around 50-90%. Passage more flasks.</td>
		</tr>
		<tr>
			<td>Cytotoxic effects of sera, i. e. due to incomplete complement-inactivation</td>
			<td>Perform a cell viability assay in addition to the mediator release assay. Increase Ag8 concentration to avoid incomplete complement-inactivation.&#160;</td>
		</tr>
		<tr>
			<td>Low signal</td>
			<td>Improve signal-to-noise ratio of the assay by diluting the 1x Tyrode&#180;s buffer in deuterium oxide (D2O) instead of dH2O, or by using a sera with higher levels of specific IgE for the allergen of interest.</td>
		</tr>
		<tr>
			<td>Allergen is not stable in Tyrode&#8217;s buffer (e. g. precipitation)&#160;</td>
			<td>Make stability tests in 1x Tyrode&#8217;s buffer prior to the assay procedure. Substitution of Tyrode&#8217;s buffer is not recommended.&#160;</td>
		</tr>
		<tr>
			<td>Problems finding the right starting concentration for the respective allergen</td>
			<td>Adaption of dilution series to cover the full release curve (more dilution steps, 1:20 dilution instead of 1:10).</td>
		</tr>
		<tr>
			<td>Poor assay performance indicated by low/no signal</td>
			<td>Avoid cytotoxic effects from either sera or antigen stimulation (e.g. enzymatic allergens). Wash and soak the cells carefully. Avoid exposure to air for too long and prevent cells from drying out.&#160;</td>
		</tr>
		<tr>
			<td>How do I know if the bottom signal plateau is reached?</td>
			<td>Add &#34;no antigen&#34; controls to your plate. These are sensitized cell, which were only stimulated with 1x Tyrode&#180;s buffer but without an allergen.</td>
		</tr>
		<tr>
			<td>Do I need a positive control in addition to the maximum lysis wells?</td>
			<td>As additional positive control a serum and antigen combination known to cause degranulation&#160; can be used or an anti-Fc&#949;R1 antibody.</td>
		</tr>
		<tr>
			<td>How many wells do I need?</td>
			<td>That depends on your titration series, the number of antigens and sera you want to analyze.&#160; Plan the layout for the 96-well plates according to how many sera/antigens you are going to test. Do not forget to add the &#34;no antigen controls&#34;, the background cells (non-sensitized, non-stimulated) as well as the maximum lysis wells.</td>
		</tr>
		<tr>
			<td>How many sera should I test? And do I need replicates?&#160;</td>
			<td>Although the assay is quite robust, there is some assay-to-assay variability due to altered cell responsiveness. Therefore, it is recommended to rather rely on biological replicates (using different sera) than on technical replicates. A minimum of eight different sera is sufficient to analyze allergens. However, as shown in Fig. 4B, significant results can already be obtained using less sera.</td>
		</tr>
	</tbody>
</table>
Table 1: Troubleshooting.

Watch this Scientific Journal Video about Humanized Mediator Release Assay as a Read-Out for Allergen Potency at JoVE.com

Humanized Mediator Release Assay as a Read-Out for Allergen Potency

Here we present the mediator release assay, using a rat basophilic leukemia cell line transfected with the human IgE receptor, to simulate the degranulation of effector cells typically observed in type 1 allergic reactions. This method investigates the biological activity of allergens in a highly sensitive, reproducible, and tailorable manner.

Mediator release assays analyze in vitro immunoglobulin E (IgE)-mediated degranulation and secretion of mediators by effector cells, such as mast cells ...

humanized-mediator-release-assay-as-a-read-out-for-allergen-potency

Research

JoVE Journal

Immunology and Infection

3.4K Views.  University of Salzburg. Here we present the mediator release assay, using a rat basophilic leukemia cell line transfected with the human IgE receptor, to simulate the degranulation of effector cells typically observed in type 1 allergic reactions. This method investigates the biological activity of allergens in a highly sensitive, reproducible, and tailorable manner.

Video: Humanized Mediator Release Assay as a Read-Out for Allergen Potency

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Murine Model of Allergen Induced Asthma

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, with the prevalence increasing.2 As with other diseases, animal models of allergic airway disease greatly facilitate understanding of the underlying pathophysiology, help identify potential therapeutic targets, and allow preclinical testing of possible new therapies. Models of allergic airway disease have been developed in several animal species, but murine models are particularly attractive due to the low cost, ready availability, and well-characterized immune systems of these animals.3 Availability of a variety of transgenic strains further increases the attractiveness of these models.4 Here we describe two murine models of allergic airway disease, both employing ovalbumin as the antigen. Following initial sensitization by intraperitoneal injection, one model delivers the antigen challenge by nebulization, the other by intratracheal delivery. These two models offer complementary advantages, with each mimicking the major features of human asthma.5
The major features of acute asthma include an exaggerated airway response to stimuli such as methacholine (airway hyperresponsiveness; AHR) and eosinophil-rich airway inflammation. These are also prominent effects of allergen challenge in our murine models,5,6 and we describe techniques for measuring them and thus evaluating the effects of experimental manipulation. Specifically, we describe both invasive7 and non-invasive8 techniques for measuring airway hyperresponsiveness as well as methods for assessing infiltration of inflammatory cells into the airways and the lung. Airway inflammatory cells are collected by bronchoalveolar lavage while lung histopathology is used to assess markers of inflammation throughout the organ. These techniques provide powerful tools for studying asthma in ways that would not be possible in humans.

Experimental mouse models of allergic asthma offer new possibilities for studying disease pathogenesis and developing new therapeutics. These models are well suited to measuring factors governing the allergic immune response, airway inflammation, and pulmonary pathophysiology.

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, ...

Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model

Small animal models such as mice have been extensively used to study human disease and to develop new therapeutic interventions. Despite the wealth of information gained from these studies, the unique characteristics of mouse immunity as well as the species specificity of viral diseases such as human immunodeficiency virus (HIV) infection led to the development of humanized mouse models. The earlier models involved the use of C. B 17 scid/scid mice and the transplantation of human fetal thymus and fetal liver termed thy/liv (SCID-hu) 1, 2 or the adoptive transfer of human peripheral blood leukocytes (SCID-huPBL) 3. Both models were mainly utilized for the study of HIV infection.
One of the main limitations of both of these models was the lack of stable reconstitution of human immune cells in the periphery to make them a more physiologically relevant model to study HIV disease. To this end, the BLT humanized mouse model was developed. BLT stands for bone marrow/liver/thymus. In this model, 6 to 8 week old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) immunocompromised mice receive the thy/liv implant as in the SCID-hu mouse model only to be followed by a second human hematopoietic stem cell transplant 4. The advantage of this system is the full reconstitution of the human immune system in the periphery. This model has been used to study HIV infection and latency 5-8.
We have generated a modified version of this model in which we use genetically modified human hematopoietic stem cells (hHSC) to construct the thy/liv implant followed by injection of transduced autologous hHSC 7, 9. This approach results in the generation of genetically modified lineages. More importantly, we adapted this system to examine the potential of generating functional cytotoxic T cells (CTL) expressing a melanoma specific T cell receptor. Using this model we were able to assess the functionality of our transgenic CTL utilizing live positron emission tomography (PET) imaging to determine tumor regression (9).
The goal of this protocol is to describe the process of generating these transgenic mice and assessing in vivo efficacy using live PET imaging. As a note, since we use human tissues and lentiviral vectors, our facilities conform to CDC NIH guidelines for Biosafety Level 2 (BSL2) with special precautions (BSL2+). In addition, the NSG mice are severely immunocompromised thus, their housing and maintenance must conform to the highest health standards (<a href="http://jaxmice.jax.org/research/immunology/005557-housing.html" target="_blank">http://jaxmice.jax.org/research/immunology/005557-housing.html</a>).

The generation and characterization of tumor specific T cells using humanized mice is described here. Human thymic tissue and genetically modified human hematopoietic stem cells are transplanted into immunocompromised mice. This results in the reconstitution of an engineered human immune system allowing for in vivo examination of anti-tumor immune responses.

Small animal models such as mice have been extensively used to study human  disease and to develop new therapeutic interventions. Despite the wealth of  ...

Humanized Mouse Model to Study Bacterial Infections Targeting the Microvasculature

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood vessels resulting in vascular leak, the development of purpuric rashes and eventual tissue necrosis. Studying the pathogenesis of this infection was previously limited by the human specificity of the bacteria, which makes in vivo models difficult. In this protocol, we describe a humanized model for this infection in which human skin, containing dermal microvessels, is grafted onto immunocompromised mice. These vessels anastomose with the mouse circulation while maintaining their human characteristics. Once introduced into this model, N. meningitidis adhere exclusively to the human vessels, resulting in extensive vascular damage, inflammation and in some cases the development of purpuric rash. This protocol describes the grafting, infection and evaluation steps of this model in the context of N. meningitidis infection. The technique may be applied to numerous human specific pathogens that infect the blood stream.

Neisseria meningitidis is a human specific pathogen that infects blood vessels. In this protocol human microvessels are introduced into a mouse by grafting human skin onto immunocompromised mice. Bacteria adhere extensively to the human vessels, leading to vascular damage and development of the purpuric rash typically observed in human cases.

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood ...

A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although a component of the innate immune response, NETs are implicated in a range of disease processes including autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay. Blood is collected using conventional venipuncture techniques. Neutrophils are isolated using dextran sedimentation and a density gradient using conditions optimized for dog blood. After allowing time for attachment to the wells of a 96 well plate, neutrophils are treated with NET-inducing agonists such as phorbol-12-myristate-13-acetate or platelet activating factor. DNA release is measured by the fluorescence of a cell-impermeable nucleic acid dye. This assay is a simple, inexpensive method for quantifying NET release, but NET formation rather than other causes of cell death must be confirmed with alternative methods.

Neutrophil extracellular traps (NETs) are networks of DNA, histones and neutrophil proteins. Although a component of the innate immune response, NETs are implicated in autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay.

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although ...

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC2010 birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e.&#160;from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. However, these screens are typically conducted in cells that are naturally permissive to the viral pathogen under study. Therefore, the robust replication of viruses in control conditions may limit the dynamic range of these screens. Furthermore, these screens may be unable to easily identify cellular defense pathways that restrict virus replication if the virus is well-adapted to the host and capable of countering antiviral defenses. In this article, we describe a new paradigm for exploring virus-host interactions through the use of screens that center on naturally abortive infections by arboviruses such as vesicular stomatitis virus (VSV). Despite the ability of VSV to replicate in a wide range of dipteran insect and mammalian hosts, VSV undergoes a post-entry, abortive infection in a variety of cell lines derived from lepidopteran insects, such as the gypsy moth (Lymantria dispar). However, these abortive VSV infections can be &#34;rescued&#34; when host cell antiviral defenses are compromised. We describe how VSV strains encoding convenient reporter genes and restrictive L. dispar cell lines can be paired to set-up screens to identify host factors involved in arbovirus restriction. Furthermore, we also show the utility of these screening tools in the identification of virally encoded factors that rescue VSV replication during coinfection or through ectopic expression, including those encoded by mammalian viruses. The natural restriction of VSV replication in L. dispar cells provides a high signal-to-noise ratio when screening for the conditions that promote VSV rescue, thus enabling the use of simplistic luminescence- and fluorescence-based assays to monitor the changes in VSV replication. These methodologies are valuable for understanding the interplay between host antiviral responses and viral immune evasion factors.

Here, we present the protocols to identify 1) virus-encoded immunomodulators that promote arbovirus replication and 2) eukaryotic host factors that restrict arbovirus replication. These fluorescence- and luminescence-based methods allow researchers to rapidly obtain quantitative readouts of arbovirus replication in simplistic assays with low signal-to-noise ratios.

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. ...

Humanized NOD/SCID/IL2r&#947;null (hu-NSG) Mouse Model for HIV Replication and Latency Studies

Ethical regulations and technical challenges for research in human pathology, immunology, and therapeutic development have placed small animal models in high demand. With a close genetic and behavioral resemblance to humans, small animals such as the mouse are good candidates for human disease models, through which human-like symptoms and responses can be recapitulated. Further, the mouse genetic background can be altered to accommodate diverse demands. The NOD/SCID/IL2r&#947;null (NSG) mouse is one of the most widely used immunocompromised mouse strains; it allows engraftment with human hematopoietic stem cells and/or human tissues and the subsequent development of a functional human immune system. This is a critical milestone in understanding the prognosis and pathophysiology of human-specific diseases such as HIV/AIDS and aiding the search for a cure. Herein, we report a detailed protocol for generating a humanized NSG mouse model (hu-NSG) by hematopoietic stem cell transplantation into a radiation-conditioned neonatal NSG mouse. The hu-NSG mouse model shows multi-lineage development of transplanted human stem cells and susceptibility to HIV-1 viral infection. It also recapitulates key biological characteristics in response to combinatorial antiretroviral therapy (cART).

This protocol provides a method to establish humanized mice (hu-NSG) via intrahepatic injection of human hematopoietic stem cells into radiation-conditioned neonatal NSG mice. The hu-NSG mouse is susceptible to HIV infection and combinatorial antiretroviral therapy (cART) and serves as a suitable pathophysiological model for HIV replication and latency investigations.

Ethical regulations and technical challenges for research in human pathology, immunology, and therapeutic development have placed small animal models in ...

Establishment of the Dual Humanized TK-NOG Mouse Model for HIV-associated Liver Pathogenesis

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of their morbidity. The liver immunopathology caused by HIV-1 remains elusive. Small xenograft animal models with human hepatocytes and human immune system can recapitulate the human biology of the disease&#39;s pathogenesis. Herein, a protocol is described to establish a dual humanized mouse model through human hepatocytes and CD34+ hematopoietic stem/progenitor cells (HSPCs) transplantation, to study liver immunopathology as observed in HIV-infected patients. To achieve dual reconstitution, male TK-NOG (NOD.Cg-Prkdcscid Il2rgtm1Sug Tg(Alb-TK)7-2/ShiJic) mice are intraperitoneally injected with ganciclovir (GCV) doses to eliminate mouse transgenic liver cells, and with treosulfan for nonmyeloablative conditioning, both of which facilitate human hepatocyte (HEP) engraftment and human immune system (HIS) development. Human albumin (ALB) levels are evaluated for liver engraftment, and the presence of human immune cells in blood detected by flow cytometry confirms the establishment of human immune system. The model developed using the protocol described here resembles multiple components of liver damage from HIV-1 infection. Its establishment could prove to be essential for studies of hepatitis virus co-infection and for the evaluation of antiviral and antiretroviral drugs.

This protocol provides a reliable method to establish humanized mice with both human immune system and liver cells. Dual reconstituted immunodeficient mice achieved via intrasplenic injection of human hepatocytes and CD34+ hematopoietic stem cells are susceptible to human immunodeficiency virus-1 infection and recapitulate liver damage as observed in HIV-infected patients.

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of ...

A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells

Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological malignancies such as childhood acute lymphocytic leukemia. Efforts are currently underway to extend this promising therapy to solid tumors in addition to other hematological cancers. Here, we describe the development and production of potent CAR T cells targeting antigens with unique or preferential expression on solid and liquid tumor cells. The in vitro potency of these CAR T cells is then evaluated in real-time using the highly sensitive impedance-based xCELLigence assay. Specifically, the impact of different costimulatory signaling domains, such as glucocorticoid-induced tumor necrosis factor receptor (TNFR)-related protein (GITR), on the in vitro potency of CAR T cells is examined. This report includes protocols for: generating CAR T cells for preclinical studies using lentiviral gene transduction, expanding CAR T cells, validating CAR expression, and running and analyzing xCELLigence potency assays.

We describe a quantitative real-time in vitro cytolysis assay system to evaluate the potency of chimeric antigen receptor T cells targeting liquid and solid tumor cells. This protocol can be extended to assess other immune effector cells, as well as combination treatments.

Humanized Mediator Release Assay as a Read-Out for Allergen Potency

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

Introducing Shear Stress in the Study of Bacterial Adhesion

Murine Model of Allergen Induced Asthma

Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model

Humanized Mouse Model to Study Bacterial Infections Targeting the Microvasculature

A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

Humanized NOD/SCID/IL2rγ^null (hu-NSG) Mouse Model for HIV Replication and Latency Studies

Establishment of the Dual Humanized TK-NOG Mouse Model for HIV-associated Liver Pathogenesis

A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells