Name: single-cell analysis of immunophenotype and cytokine production in peripheral whole blood via mass cytometry
Uploaded: 2018-06-26T13:30:00.000+00:00
Duration: 12 min 36 s
Description: Watch this Scientific Journal Video about Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry at JoVE.com

We would like to thank Aimee Pugh-Bernard for her intellectual input and helpful comments. This work was supported by the Boettcher Foundation Webb-Waring Biomedical Research Award and award number K23-1K23AR070897 from the NIH to Elena W.Y. Hsieh. She was also supported by award number K12-HD000850 from the Eunice Kennedy Shriver National Institute of Child Health and Human Development and the Lucile Packard Foundation for Children&#39;s Health, Stanford CTSA UL1 TR001085, and Child Health Research Institute of Stanford University.

All methods described here have been approved by the Colorado Multiple Institutional Review Board (COMIRB) of the University of Colorado. All described procedures below should be performed in a sterile tissue culture hood unless stated otherwise, with filtered pipette tips, and all reagents filtered.
1. Preparation of Reagents for Peripheral Whole Blood Processing
<ol>
	<li>Prepare ruxolitinib stock aliquots (10&#8211;15 &#956;L/vial for single use) at 10 mM by diluting the lyophilized reagent in DMSO as per the manufacturer&#8217;s instructions (store at -80 &#176;C). Keep DMSO concentrations in all assays including unstimulated controls below 0.2% (vol/vol).</li>
	<li>Prepare R848 (resiquimod) stock aliquots (10&#8211;15 &#956;L/vial for single use) at 1 &#956;g/&#956;L by diluting the lyophilized reagent in sterile water as the per manufacturer&#8217;s instructions. Store at -80 &#176;C.</li>
	<li>Prepare lipopolysaccharide (LPS) stock aliquots (5 &#956;L per vial for one time use only) at 1 &#956;g/&#956;L by diluting the lyophilized reagent in sterile water as per manufacturer instructions. Store at -80 &#176;C.</li>
	<li>Prepare the Cell Staining Media (CSM) using PBS, 0.5% BSA, and 0.02% NaN3.</li>
	<li>Acquire and keep ready the protein transport inhibitor (PTI) cocktail (Table of Materials).</li>
	<li>Thaw and dilute the ruxolitinib stock vial (10 mM) 1:10 in sterile PBS (1 mM). Set aside at room temperature in a tissue culture hood.</li>
	<li>Thaw and dilute the R848 stock vial (1 &#956;g/&#181;L) 1:10 in sterile PBS (0.1 &#956;g/&#181;L). Set aside at room temperature in a tissue culture hood.</li>
	<li>Thaw and dilute the LPS stock vial (1 &#956;g/&#181;L) 1:100 in sterile PBS (0.01 &#956;g/&#181;L). Set aside at room temperature in a tissue culture hood.</li>
	<li>Pre-warm sterile RPMI (no L-glutamine) in a standard 37 &#176;C water bath (for at least 10 min).</li>
	<li>Dilute the lyse/fix buffer by 1:5 in filtered ddH2O (working concentration) on the benchtop (does not need to be sterile). 
	Note: 1 mL of whole blood requires 20 mL of working concentration of lyse/fix buffer.
	<ol>
		<li>Make lyse/fix buffer for 5 conditions of 1 mL of whole blood each (at least 100 mL). Aliquot 20 mL of the working concentration of lyse/fix buffer into 50 mL conical tubes per milliliter of whole blood (keep the lyse/fix solution at 37 &#176;C until needed). Label the conditions on the conical tubes containing lyse/fix buffer as follows: T0 (Time zero), T6 + 5R (Time 6 h with 5 uM ruxolitinib), T6 + R848 (Time 6 h with 1 &#956;g/mL R848), T6 + LPS (Time 6 h with 0.1 &#956;g/mL LPS), T6 (Time 6 h).</li>
	</ol>
	</li>
	<li>Label round bottom sterile polystyrene tubes with caps and 1 mL microcentrifuge tubes with the same nomenclature as in step 1.10.</li>
</ol>
2. Stimulation and Processing of Peripheral Whole Blood (Figure 1)
<ol>
	<li>Place the labeled round bottom polystyrene tubes from step 1.11 (T0, T6 + 5R, T6 + R848, T6 + LPS, T6) on a rack. 
	Note: All the following steps are performed in this type of tube unless specified otherwise. Cap the tubes when transferring between the tissue culture hood and incubator to maintain sterility.</li>
	<li>Add 1 mL of 37 &#176;C RPMI (no L-glutamine) to each of the tubes (T0, T6 + 5R, T6 + R848, T6 + LPS, T6). Invert the blood collection tube several times, add 1 mL of whole blood to each tube, and pipette up and down several times to mix thoroughly with RPMI (dilution with RPMI prevents clumping of the blood during the 6 h incubation period).</li>
	<li>Add 10 &#956;L of the 1:10 ruxolitinib to T6 + 5R. Mix thoroughly with a P1000 pipette. Place the rack of tubes in the incubator at 37 &#176;C and begin timing the incubation (T = 0).</li>
	<li>Process the T0 sample as follows.
	<ol>
		<li>Pipette the entire contents of the T0 tube into a labeled conical tube containing 20 mL of 37 &#176;C working concentration lyse/fix buffer. To optimize cell recovery, rinse the tube with working concentration lyse/fix buffer. Mix by inverting the conical tube.</li>
		<li>Incubate at 37 &#176;C for 15 min to allow for lysis and fixation. Following fixation, perform all subsequent steps at the benchtop. Centrifuge cells at 500 x g for 5 min at room temperature.</li>
		<li>Decant the supernatant. Resuspend the cells in 1 mL of ice cold PBS to break up the pellet, then fill the conical to a 15 mL volume with PBS.</li>
		<li>Centrifuge the cells at 500 x g for 5 min at room temperature. Decant the supernatant. Repeat the PBS wash (steps 2.4.3&#8211;2.4.4) if pellets are red. Resuspend the cells in 1 mL of CSM to break up the pellet, then transfer to the labeled microcentrifuge tube (step 1.11) for antibody staining. Take a 10 &#181;L sample to count cells using an automated cell counter or hemocytometer. 
		Note: Since all cells are fixed at this point, it is not necessary to include a viability stain.</li>
		<li>Centrifuge the cells at 500 x g for 5 min at room temperature. Aspirate the supernatant, leaving the pellet in ~60 &#181;L of residual volume. Keep this pellet on ice until samples from other conditions have completed processing.</li>
	</ol>
	</li>
	<li>At T = 30 min, move the tube rack (step 2.3) to the tissue culture hood and perform the following.
	<ol>
		<li>Add 10 &#956;L of the 0.1 &#956;g/&#956;L R848 to T6 + R848. Mix thoroughly with a P1000 pipette.</li>
		<li>Add 10 &#956;L of the 0.01 &#956;g/&#956;L LPS to T6 + LPS. Mix thoroughly with a P1000 pipette.</li>
		<li>Add 4 &#956;L of protein transport inhibitor (stock at 500X) to T6, T6 + 5R, and T6 + LPS (but NOT to T6 + R848) and return the samples to the incubator until T = 6 h. Mix thoroughly with a P1000 pipette every 2 h. 
		Note: R848 is an endoplasmic TLR agonist and therefore requires a temporal delay in the addition of the protein transport inhibitor to allow for access to its target receptor.</li>
	</ol>
	</li>
	<li>At T = 2 h, add 4 &#956;L of protein transport inhibitor cocktail (stock at 500X) to the T6 + R848 tube. Mix all samples with a P1000 pipette and return the rack to the incubator until T = 6 h.</li>
	<li>Mix the samples with a P1000 one more time at T = 4 h.</li>
	<li>At T = 6 h, process T6, T6 + LPS, T6 + R848, and T6 + 5R blood sample tubes as described in step 2.4 for the T0 sample.</li>
	<li>Store the cell pellets in residual CSM volume at -80 &#176;C to process on a future date. Alternatively, proceed to barcoding and antibody staining without storing.</li>
</ol>
3. Barcode of Lysed/Fixed Blood Cells
<ol>
	<li>Thaw the lysed/fixed cell samples from the -80 &#176;C storage slowly on ice; a maximum of 20 samples can be labeled with unique barcodes and pooled using this system. Dilute the 10X barcoding perm buffer by 1:10 with PBS; make enough buffer for ~3 mL per sample.</li>
	<li>Fill one non-sterile trough with the CSM and one with the 1X barcoding perm buffer. Add 1 mL of ice cold CSM to freshly thawed samples, mix thoroughly with a pipette, and transfer to the respective pre-labeled polypropylene cluster tubes.</li>
	<li>Take a 10 &#956;L sample to count cells using an automated cell counter or hemocytometer. Normalize the cell counts to 1.5&#8211;2 x 106 cells per sample in each cluster tube: remove and discard the volume of excess cells above 2 x 106 cells.</li>
	<li>Centrifuge the cells at 600 x g for 5 min at room temperature. Resuspend the cells in 1 mL of 1X barcoding perm buffer with a multichannel pipette and centrifuge at 600 x g for 5 min at room temperature. Aspirate off the supernatant.</li>
	<li>Line up the cluster tubes on a rack in the same order as indicated on the barcode key so the sample matches with its barcode. Add 800 &#956;L 1X barcoding perm buffer by multichannel pipette to all samples in the cluster tubes without touching the cell pellet with the pipette tips (no mixing) to reduce cell loss. Set aside the rack with the cluster tubes.</li>
	<li>Remove 20-plex Pd Barcoding Kit tube strips from -20 &#176;C and thaw at room temperature. Add 100 &#956;L of 1X barcoding perm buffer, mix thoroughly, and transfer 120 &#956;L of the resuspended barcode mix into the corresponding cell samples in the cluster tubes.</li>
	<li>Mix thoroughly by multichannel pipette so that there is no cross-contamination between individually barcoded samples. Incubate cluster tubes for 30 min at room temperature to allow the barcodes to label the cells.</li>
	<li>Centrifuge at 600 x g for 5 min at room temperature. Aspirate the supernatant, then resuspend in 1 mL of CSM.</li>
	<li>Centrifuge and resuspend in CSM again as in step 3.8. 
	Note: Each sample is now labeled with a unique barcode, and samples are ready to be pooled.</li>
	<li>Centrifuge at 600 x g for 5 min at room temperature and aspirate the supernatant. With a single pipette and using the same tip, transfer all cell pellets in ~70&#8211;80 &#956;L residual volume to one polystyrene tube. Do NOT eject the pipette tip; set aside the single pipette with this tip.</li>
	<li>With a multichannel pipette and new tips, add ~100 &#956;L CSM to each original cluster tube to maximize cell recovery. With the single pipette with the tip that was set aside, transfer all cell pellets in ~100 &#956;L residual volume to the same polystyrene tube.</li>
	<li>Add CSM to top off the polystyrene tube (~3 mL). Count and record the cell number of the pooled barcode set. Centrifuge at 600 x g for 5 min at room temperature and aspirate the supernatant. Proceed to staining the barcoded samples on the same day. 
	Note: It is normal to expect ~20&#8211;30% cell loss in the barcoding process.</li>
</ol>
4. Staining of Barcoded Lysed/Fixed Blood Cells and Preparation for Analysis on Mass Cytometry Instrument
Note: Each 1X titer of staining antibody (1 &#956;L of antibody per 100 &#956;L staining reaction), can usually stain 3&#8211;4 x 106 cells. Therefore, when all barcoded samples are pooled into one tube, the amount of antibody must be scaled up. If 20 barcoded samples amount to 30 x 106 cells, and each 1X titer can stain 3&#8211;4 x 106 cells, the barcoded sample only requires a 10X titer, as opposed to staining each sample individually, which would require a 20X amount of antibody (1X per individual tube). The concentration of the antibody to cell number should be carefully titrated for each individual antibody cocktail (not discussed here).
<ol>
	<li>Stain the cells using antibodies (Table of Materials) for surface staining and cytokine induction.
	<ol>
		<li>Make the surface staining cocktail by calculating the amount to be added and accounting for pipetting error (i.e., if staining 20 barcoded samples of 2 x 106 cells in each sample, a 10X titer stain is required; for final volume calculations, compensate for pipetting error by making a 10.5X final staining solution).</li>
		<li>Add 10X worth of surface staining volume to the barcoded cell pellet. To reduce cell loss, with the same tip, mix and measure up the volume of surface stains and cell pellet.</li>
		<li>Based on the volume of cells and staining cocktail, add CSM to a final staining volume of 50 &#956;L per number of cell samples (i.e., if running a set of 20 samples, 50 &#956;L X 20 = 1 mL). Incubate for 30 min at 4 &#176;C. Agitate the sample every 15 min to promote even staining.</li>
		<li>During the surface stain incubation, prepare the Perm/Wash buffer (Table of Materials) by diluting 1:10 with filtered ddH2O. Prepare volumes of 2 mL for permeabilization, and 5 mL for washing. Keep at 4 &#176;C or on ice.</li>
		<li>Top off the surface staining tube with CSM, and centrifuge at 600 x g at 4 &#176;C for 5 min. Aspirate the supernatant. Resuspend the barcoded sample in 2 mL of 4 &#176;C, 1:10 dilution Perm/Wash buffer. Incubate at 4 &#176;C for 20&#8211;30 min to fully permeabilize the cells.</li>
		<li>Centrifuge at 600 x g at 4 &#176;C for 5 min and aspirate the supernatant.</li>
		<li>For intracellular staining, follow similar staining steps (as for surface staining). However, use the 4 &#176;C, 1:10 dilution Perm/Wash buffer instead of the CSM to make the total staining volume so that the cells remain in a permeabilizing environment throughout the intracellular staining.</li>
		<li>Add the intracellular antibody cocktail to the surface stained and permeabilized cell pellet (steps 4.1.2&#8211;4.1.17). Bring the total staining volume to 50 &#956;L per number of cell samples. Incubate for 60 min at 4 &#176;C. Agitate the sample every 15 min to ensure even staining.</li>
		<li>During the intracellular staining, prepare the intercalator solution: 900 &#956;L of filtered PBS + 100 &#956;L of filtered 16% PFA (final concentration of 1.6% PFA) + 0.2 &#956;L of 500 uM of intercalator dye (Table of Materials).</li>
		<li>Top off the cell pellet + intracellular antibody cocktail with cold 1:10 Perm/Wash buffer.</li>
		<li>Centrifuge at 600 x g at 4 &#176;C for 5 min, and aspirate the supernatant. Top off the staining tube with CSM. Count and record the cell number. Centrifuge at 600 x g at 4 &#176;C for 5 min, and aspirate the supernatant. Resuspend the cells in 1 mL of intercalator solution from step 4.9. Incubate for at least 20 min at room temperature for full intercalation, or overnight at 4 &#176;C (samples in intercalator solution can stay at 4 &#176;C for up to 1 week before running them on the mass cytometry instrument).</li>
	</ol>
	</li>
	<li>Prepare the cells for the mass cytometry instrument.
	<ol>
		<li>Top off the tube with 3 mL of filtered ddH2O, and centrifuge at 600 x g for 5 min at 4 &#176;C. Resuspend the cells in 3 mL of filtered ddH2O. Pass this suspension through a 100 &#956;m filter to remove any debris or aggregates that could potentially clog the mass cytometry instrument.</li>
		<li>Count and record the cell number post-filtration. Centrifuge at 600 x g for 5 min at 4 &#176;C</li>
	</ol>
	</li>
	<li>Prepare the calibration bead solution (Table of Materials) by diluting it 1:10 in filtered ddH2O.</li>
	<li>Resuspend the stained cells in the required volume of 1:10 diluted calibration bead solution to attain a cell concentration of ~1 x 106 cells/mL. 
	Note: For a CyTOF1 at 45 &#181;L/min, the recommended optimum is 5 x 105 cells/mL; for Helios at 30 &#181;L/min, 7.5 x 105 cells/mL.</li>
	<li>Proceed to run the sample on the mass cytometry instrument9.</li>
</ol>

<ol>
	<li>Walsh, S. J., Rau, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoimmune+diseases:+a+leading+cause+of+death+among+young+and+middle-aged+women+in+the+United+States.">Autoimmune diseases: a leading cause of death among young and middle-aged women in the United States.</a> American journal of public health. 90 (9), 1463-1466 (2000).</li><li>Mak, A., Cheung, M. W. -. L., Chiew, H. J., Liu, Y., Ho, R. C. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+trend+of+survival+and+damage+of+systemic+lupus+erythematosus:+meta-analysis+and+meta-regression+of+observational+studies+from+the+1950s+to+2000s.">Global trend of survival and damage of systemic lupus erythematosus: meta-analysis and meta-regression of observational studies from the 1950s to 2000s.</a> Seminars in arthritis and rheumatism. 41 (6), 830-839 (2012).</li><li>Kaul, A., Gordon, C., 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=Systemic+lupus+erythematosus.">Systemic lupus erythematosus.</a> Nature reviews. Disease primers. 2, 16039 (2016).</li><li>Annunziato, F., Romagnani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+human+effector+CD4++T+cells.">Heterogeneity of human effector CD4+ T cells.</a> Arthritis Research &amp; Therapy. 11 (6), 257 (2009).</li><li>Peck, A., Mellins, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+of+T-cell+phenotype+and+function:+the+T+helper+type+17+example.">Plasticity of T-cell phenotype and function: the T helper type 17 example.</a> Immunology. 129 (2), 147-153 (2010).</li><li>Basso, A. S., Cheroutre, H., Mucida, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=More+stories+on+Th17+cells.">More stories on Th17 cells.</a> Cell research. 19 (4), 399-411 (2009).</li><li>Ornatsky, O., Bandura, D., Baranov, V., Nitz, M., Winnik, M. A., Tanner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+multiparametric+analysis+by+mass+cytometry.">Highly multiparametric analysis by mass cytometry.</a> Journal of Immunological Methods. 361 (1-2), 1-20 (2010).</li><li>Ornatsky, O., Baranov, V. I., Bandura, D. R., Tanner, S. D., Dick, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+cellular+antigen+detection+by+ICP-MS.">Multiple cellular antigen detection by ICP-MS.</a> Journal of Immunological Methods. 308 (1-2), 68-76 (2006).</li><li>Bendall, S. C., Simonds, E. F., 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=Single-Cell+Mass+Cytometry+of+Differential+Immune+and+Drug+Responses+Across+a+Human+Hematopoietic+Continuum.">Single-Cell Mass Cytometry of Differential Immune and Drug Responses Across a Human Hematopoietic Continuum.</a> Science. 332 (6030), 687-696 (2011).</li><li>Bandura, D. R., Baranov, V. I., 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=Mass+Cytometry:+Technique+for+Real+Time+Single+Cell+Multitarget+Immunoassay+Based+on+Inductively+Coupled+Plasma+Time-of-Flight+Mass+Spectrometry.">Mass Cytometry: Technique for Real Time Single Cell Multitarget Immunoassay Based on Inductively Coupled Plasma Time-of-Flight Mass Spectrometry.</a> Analytical Chemistry. 81 (16), 6813-6822 (2009).</li><li>Leipold, M. D., Maecker, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+cytometry:+protocol+for+daily+tuning+and+running+cell+samples+on+a+CyTOF+mass+cytometer.">Mass cytometry: protocol for daily tuning and running cell samples on a CyTOF mass cytometer.</a> Journal of visualized experiments : JoVE. (69), e4398 (2012).</li><li>McCarthy, R. L., Duncan, A. D., Barton, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+Preparation+for+Mass+Cytometry+Analysis.">Sample Preparation for Mass Cytometry Analysis.</a> Journal of visualized experiments : JoVE. (122), (2017).</li><li>Rahman, A., Isenberg, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+lupus+erythematosus.">Systemic lupus erythematosus.</a> The New England journal of medicine. 358 (9), 929-939 (2008).</li><li>O'Gorman, W. E., Hsieh, E. W. 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=Single-cell+systems-level+analysis+of+human+Toll-like+receptor+activation+defines+a+chemokine+signature+in+patients+with+systemic+lupus+erythematosus.">Single-cell systems-level analysis of human Toll-like receptor activation defines a chemokine signature in patients with systemic lupus erythematosus.</a> The Journal of allergy and clinical immunology. 136 (5), 1326-1336 (2015).</li><li>O'Gorman, W. E., Kong, D. 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=Mass+cytometry+identifies+a+distinct+monocyte+cytokine+signature+shared+by+clinically+heterogeneous+pediatric+SLE+patients.">Mass cytometry identifies a distinct monocyte cytokine signature shared by clinically heterogeneous pediatric SLE patients.</a> Journal of Autoimmunity. 81, 74-89 (2017).</li><li>Simonds, E. F., Bendall, S. C., 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=Extracting+a+cellular+hierarchy+from+high-dimensional+cytometry+data+with+SPADE.">Extracting a cellular hierarchy from high-dimensional cytometry data with SPADE.</a> Nature Biotechnology. , 1-8 (2011).</li><li>Amir, E. -. A. D., Davis, K. 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=viSNE+enables+visualization+of+high+dimensional+single-cell+data+and+reveals+phenotypic+heterogeneity+of+leukemia.">viSNE enables visualization of high dimensional single-cell data and reveals phenotypic heterogeneity of leukemia.</a> Nature Biotechnology. 31 (6), 545-552 (2013).</li><li>Bruggner, R. V., Bodenmiller, B., Dill, D. L., Tibshirani, R. J., Nolan, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+identification+of+stratifying+signatures+in+cellular+subpopulations.">Automated identification of stratifying signatures in cellular subpopulations.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (26), E2770-E2777 (2014).</li><li>Levine, J. H., Simonds, E. F., 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=Data-Driven+Phenotypic+Dissection+of+AML+Reveals+Progenitor-like+Cells+that+Correlate+with+Prognosis.">Data-Driven Phenotypic Dissection of AML Reveals Progenitor-like Cells that Correlate with Prognosis.</a> Cell. 162 (1), 184-197 (2015).</li><li>Samusik, N., Good, Z., Spitzer, M. H., Davis, K. L., Nolan, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+mapping+of+phenotype+space+with+single-cell+data.">Automated mapping of phenotype space with single-cell data.</a> Nature Publishing Group. 13 (6), 493-496 (2016).</li><li>Spitzer, M. H., Gherardini, P. F., 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=IMMUNOLOGY.+An+interactive+reference+framework+for+modeling+a+dynamic+immune+system.">IMMUNOLOGY. An interactive reference framework for modeling a dynamic immune system.</a> Science. 349 (6244), 1259425 (2015).</li><li>Fienberg, H. G., Simonds, E. F., Fantl, W. J., Nolan, G. P., Bodenmiller, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+platinum-based+covalent+viability+reagent+for+single-cell+mass+cytometry.">A platinum-based covalent viability reagent for single-cell mass cytometry.</a> Cytometry Part A. 81 (6), 467-475 (2012).</li><li>Jansen, K., Blimkie, D., 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=Polychromatic+flow+cytometric+high-throughput+assay+to+analyze+the+innate+immune+response+to+Toll-like+receptor+stimulation.">Polychromatic flow cytometric high-throughput assay to analyze the innate immune response to Toll-like receptor stimulation.</a> Journal of Immunological Methods. 336 (2), 183-192 (2008).</li><li>Zunder, E. R., Finck, 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=Palladium-based+mass+tag+cell+barcoding+with+a+doublet-filtering+scheme+and+single-cell+deconvolution+algorithm.">Palladium-based mass tag cell barcoding with a doublet-filtering scheme and single-cell deconvolution algorithm.</a> Nature Protocols. 10 (2), 316-333 (2015).</li><li>Lai, L., Ong, R., Li, J., Albani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+CD45-based+barcoding+approach+to+multiplex+mass-cytometry+(CyTOF).">A CD45-based barcoding approach to multiplex mass-cytometry (CyTOF).</a> Cytometry Part A. , (2015).</li><li>Mei, H. E., Leipold, M. D., Schulz, A. R., Chester, C., Maecker, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Barcoding+of+live+human+peripheral+blood+mononuclear+cells+for+multiplexed+mass+cytometry.">Barcoding of live human peripheral blood mononuclear cells for multiplexed mass cytometry.</a> The Journal of Immunology. 194 (4), 2022-2031 (2015).</li><li>Finck, R., Simonds, E. F., 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=Normalization+of+mass+cytometry+data+with+bead+standards.">Normalization of mass cytometry data with bead standards.</a> Cytometry Part A. 83 (5), 483-494 (2013).</li><li>Takahashi, C., Au-Yeung, 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=Mass+cytometry+panel+optimization+through+the+designed+distribution+of+signal+interference.">Mass cytometry panel optimization through the designed distribution of signal interference.</a> Cytometry. Part A : the journal of the International Society for Analytical Cytology. 91 (1), 39-47 (2017).</li></ol>

The authors have nothing to disclose.

Here we describe a novel, single-cell, proteomic approach to simultaneously assess multiple immune cell types and detect their various cytokine perturbations in the milieu of patient specific &#34;pathogenic&#34; peripheral blood plasma circulating factors. This method employs peripheral whole blood as the analytical vehicle, and mass cytometry as the tool for the evaluation of immune cellular phenotypic and functional abnormalities. The method is readily applicable to human and mice studies21, an...

Autoimmune diseases are a major cause of morbidity and mortality affecting 3-8% of the population. In the United States, autoimmune disorders are among the leading causes of death among young and middle-aged women (ages &#60;65 years)1,2. Autoimmune disorders are characterized by heterogeneous clinical presentation and diverse underlying immunological processes. The spectrum of heterogeneity is well represented across different disorders, such as joint involvement in rheumatoid arthritis (RA) and neurological disease in multiple sclerosis (MS). However, this level of heterogeneity is also exemplified within a single disorder, such as systemic lupus erythematosus (SLE): some patients may present with renal pathology, while others suffer from hematologic or joint involvement3.
The underlying immunopathogenesis in autoimmune disorders mirrors the clinical heterogeneity, involving auto- and hyper-activation of multiple innate and adaptive immune cell subsets, and concomitant dysregulated cytokine production. While cytokines play a pivotal role in the pathogenesis of autoimmune disease, understanding their specific role in the mechanism of disease has proven to be challenging. Cytokines are characterized by pleiotropy (one cytokine can have multiple effects on different cell types), redundancy (multiple cytokines can have the same effect), duality (one cytokine can have pro- or anti-inflammatory effects under different conditions), and plasticity (cytokines can be molded into a role different from its &#34;original&#34; one, depending on the environment)4,5,6. Consequently, population-level methods cannot distinguish heterogeneous cellular responses to the same &#34;cytokine milieu&#34;. Similarly, study designs that focus on one specific aspect of the immune system (a single cell type or cytokine), do not offer a global assessment of all the elements involved in complex autoimmune disease. While patient sera-based studies have afforded important insights into autoimmunity, they do not provide the specific cellular source of the dysregulated cytokines detected.
Recently, we developed a single-cell proteomic approach to simultaneously assess multiple immune cell types, and detect their various cytokine perturbations in the milieu of patient specific &#34;pathogenic&#34; peripheral blood plasma circulating factors. The workflow described here is characterized by the use of intact peripheral whole blood samples, as opposed to isolated peripheral blood mononuclear cells (PBMCs). Peripheral whole blood represents the most physiologically relevant vehicle to study systemic immune-mediated disease, including 1) non-mononuclear blood cells often involved in autoimmune disease (i.e., neutrophils, platelets), and 2) plasma circulating factors, such as nucleic acids, immune complexes, and cytokines, which have immune activating roles. To capture the &#34;intrinsic pathogenic&#34; dysregulated cytokine production, peripheral blood samples are processed immediately after the blood draw (T0, Time zero), and after 6 h of incubation at 37 &#176;C (physiological body temperature) with a protein transport inhibitor in the absence of any exogenous stimulating condition (T6, Time 6 h), to detect cytokine production (accumulation, T6-T0) that would reflect the &#34;intrinsic&#34; disease state (Figure 1). To study dysregulated processes that reflect over or under-activation of signaling pathways involved in immune responses relevant to the disease, peripheral blood samples are treated (6 h incubation at 37 &#176;C with a protein transport inhibitor) with an exogenous stimulating condition that reflects disease pathogenesis, such as Toll-Like-Receptor (TLR) agonists in the case of SLE (T6 + R848, Time 6 h with 1 &#181;g/mL R848), to detect cytokine production that would reflect a response to nucleic acids (comparing T0 vs. T6 vs. T6 + R848, Figure 1). To study immunomodulatory effects of available therapeutics ex vivo, as they pertain to the precise immune dysregulated processes for specific patients, peripheral blood samples are treated with a JAK inhibitor at the relevant therapeutic concentration (here, 5 uM ruxolitinib; T6 + 5R, Time 6 h with 5 uM ruxolitinib), to detect changes in &#34;intrinsic&#34; disease state in response to the drug (T0 vs. T6 vs. T6 + 5R, Figure 1). A JAK inhibitor was chosen for this study because JAK inhibitors have been shown to be successful in the treatment of autoimmune disorders such as RA.
To simultaneously evaluate the dysregulated processes described above in multiple immune cell subsets, peripheral blood samples from SLE patients and healthy controls were processed as described above and analyzed by mass cytometry. Mass cytometry, also known as Cytometry-Time-Of-Flight, offers single-cell analysis of over 40 parameters without issues of spectral overlap7,8,9. This technique utilizes rare earth metal isotopes in the form of soluble metal ions as tags bound to antibodies, instead of fluorophores10. Additional details regarding the mass cytometry technological platform (i.e., tuning and calibration, sample acquisition) can be found in Leipold et al. and McCarthy et al.11,12 The high-dimensionality of mass cytometry enables simultaneous measurement of multiple cytokines throughout innate and adaptive immune cell subsets with single-cell granularity (Table of Materials).
Current conventional clinical and laboratory parameters are often not sensitive or specific enough for detecting ongoing disease activity or the response to specific immunomodulators13, reflecting the need to better delineate the underlying immune changes that drive flare-ups. Given the pervasiveness of cytokine dysregulation in autoimmune disease, a plethora of treatment approaches that use antibodies or small molecular inhibitors targeting cytokines or signaling proteins involved in the regulation of cytokine production have recently emerged. In its basic format, the peripheral blood analytical approach described here provides a platform to identify patient-specific dysregulated cell subsets and their abnormal cytokine production in autoimmune disease with systemic manifestations. This methodology allows for the personalization of therapeutic choices as specific dysregulated cytokines can be identified, and specific treatment options can be tested ex vivo to assess their ability to immunomodulate the patient specific disease process.

<table><tbody><tr><td>Ruxolitinib</td><td>Santa Cruz</td><td>SC-364729A</td><td>Stock Conc: 10 mM; Final Conc: 5 &amp;mu;M</td></tr><tr><td>R848</td><td>Invivogen</td><td>tlrl-r848-5</td><td>Stock Conc: 1 &amp;mu;g/&amp;mu;L; Final Conc: 1 &amp;mu;g/mL</td></tr><tr><td>LPS-EK</td><td>Invivogen</td><td>tlrl-eklps</td><td>Stock Conc: 1 &amp;mu;g/&amp;mu;L; Final Conc: 0.1 &amp;mu;g/mL</td></tr><tr><td>Sterile PBS</td><td>Lonza</td><td>17-516F</td><td></td></tr><tr><td>Lyse/Fix Buffer</td><td>BD biosciences</td><td>558049</td><td>Stock Conc: 5X; Final Conc: 1X (dilute in ddH2O)</td></tr><tr><td>BD Phosflow perm/wash buffer I</td><td>BD biosciences</td><td>557885</td><td>Stock Conc: 10X; Final Conc: 1:10 (dilute in ddH2O)</td></tr><tr><td>RPMI</td><td>Gibco</td><td>21870-076</td><td></td></tr><tr><td>Sodium Azide (NaN3)</td><td>Sigma-Aldrich</td><td>S-8032</td><td>Stock Conc: &gt;99.9%; Final Conc: 0.0002</td></tr><tr><td>Protein Transport Inhibitor (PTI)</td><td>eBiosciences</td><td>00-4980-93</td><td>Stock Conc: 500X; Final Conc: 1X</td></tr><tr><td>DNA Intercalator</td><td>Fluidigm</td><td>201192B</td><td>Stock Conc: 500 &amp;mu;M; Final Conc: 0.1 &amp;mu;M</td></tr><tr><td>Cell Staining Media (CSM)</td><td></td><td></td><td>PBS + 0.5% BSA, 0.02% NaN3</td></tr><tr><td>MaxPar Barcode Perm Buffer</td><td>Fluidigm</td><td>201057</td><td>Stock Conc: 10X; Final Conc: 1X</td></tr><tr><td>20-plex Pd Barcode Set</td><td>Fluidigm</td><td>S0014</td><td>Stock Conc: n/a; Final Conc: n/a</td></tr><tr><td>EQ &lt;sup&gt;TM&lt;/sup&gt; Four Element Calibration Beads</td><td>Fluidigm</td><td>201078</td><td>Stock Conc: 10X; Final Conc: 1X</td></tr><tr><td>16% MeOH-free Formaldehyde Solution</td><td>Thermo</td><td>28908</td><td>Stock Conc: 16% (w/v); Final Conc: 1.6% (w/v)</td></tr><tr><td>Sterile round bottom polystyrene tubes</td><td>VWR</td><td>60818-496</td><td>Stock Conc: n/a; Final Conc: n/a</td></tr><tr><td>Polypropylene cluster tubes</td><td>Light Labs</td><td>A-9001</td><td>Stock Conc: n/a; Final Conc: n/a</td></tr><tr><td>Helios CyTOF instrument</td><td>Fluidigm</td><td>Helios</td><td>All solutions to be used in CyTOF analysis need to be free of metal contamination. ddH2O is used in the preparation of any solutions should have a resistivity of at least 18.0 M&amp;Omega;.cm. ddH2O and any self-prepared solutions should be stored in new plastic or glass bottles that have never been autoclaved.</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Antibodies used for Mass Cytometry&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Surface markers&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CD1c</td><td>Biolegend</td><td>L161</td><td>Mass: 161</td></tr><tr><td>CD3</td><td>BD</td><td>UCHT1</td><td>Mass: 144</td></tr><tr><td>CD4</td><td>Biolegend</td><td>SK3</td><td>Mass: 174</td></tr><tr><td>CD7</td><td>BD</td><td>M-T701</td><td>Mass: 149</td></tr><tr><td>CD8</td><td>Biolegend</td><td>SK1</td><td>Mass: 142</td></tr><tr><td>CD11b</td><td>Fluidigm</td><td>ICRF44</td><td>Mass: 209</td></tr><tr><td>CD11c</td><td>BD</td><td>B-ly6</td><td>Mass: 152</td></tr><tr><td>CD15</td><td>BD</td><td>HI98</td><td>Mass: 115</td></tr><tr><td>CD14</td><td>Biolegend</td><td>M5E2</td><td>Mass: 154</td></tr><tr><td>CD16</td><td>eBioscience/Thermo</td><td>B73.1</td><td>Mass: 165</td></tr><tr><td>CD19</td><td>Santa Cruz</td><td>SJ25C1</td><td>Mass: 163</td></tr><tr><td>CD21</td><td>Biolegend</td><td>Bu32</td><td>Mass: 141</td></tr><tr><td>CD27</td><td>BD</td><td>L128</td><td>Mass: 155</td></tr><tr><td>CD38</td><td>Fluidigm</td><td>HIT2</td><td>Mass: 172</td></tr><tr><td>CD45 total</td><td>Biolegend</td><td>HI30</td><td>Mass: 89</td></tr><tr><td>CD45RA</td><td>Biolegend</td><td>HI100</td><td>Mass: 153</td></tr><tr><td>CD56</td><td>Miltenyi</td><td>REA196</td><td>Mass: 168</td></tr><tr><td>CD66</td><td>BD</td><td>B1.1/CD66</td><td>Mass: 113</td></tr><tr><td>CD86</td><td>Fluidigm</td><td>IT2.2</td><td>Mass: 150</td></tr><tr><td>CD123</td><td>Fluidigm</td><td>6H6</td><td>Mass: 143</td></tr><tr><td>CD278/ICOS</td><td>Biolegend</td><td>C398.4A</td><td>Mass: 156</td></tr><tr><td>CD179/PD1</td><td>Biolegend</td><td>EH12.2H7</td><td>Mass: 162</td></tr><tr><td>IgD</td><td>Biolegend</td><td>IA6-2</td><td>Mass: 146</td></tr><tr><td>IgM</td><td>Biolegend</td><td>MHM-88</td><td>Mass: 151</td></tr><tr><td>CXCR5</td><td>BD</td><td>RF8B2</td><td>Mass: 173</td></tr><tr><td>HLADR</td><td>Biolegend</td><td>L243</td><td>Mass: 167</td></tr><tr><td>&lt;strong&gt;Cytokines&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>IL-1&amp;alpha;</td><td>Biolegend</td><td>364-3B3-14</td><td>Mass: 147</td></tr><tr><td>IL-1&amp;beta;</td><td>Biolegend&amp;nbsp;</td><td>H1b-98</td><td>Mass: 169</td></tr><tr><td>IL-1RA</td><td>Santa Cruz&amp;nbsp;</td><td>AS17</td><td>Mass: 157</td></tr><tr><td>IL-6</td><td>Biolegend</td><td>MQ2-13A5</td><td>Mass: 164</td></tr><tr><td>IL-8</td><td>BD</td><td>E8N1</td><td>Mass: 160</td></tr><tr><td>IL-12/IL-23p40</td><td>Biolegend&amp;nbsp;</td><td>C8.6</td><td>Mass: 171</td></tr><tr><td>IL-17A</td><td>Biolegend&amp;nbsp;</td><td>BL168</td><td>Mass: 148</td></tr><tr><td>IL23p19</td><td>eBioscience/Thermo</td><td>23dcdp</td><td>Mass: 176</td></tr><tr><td>MIP1&amp;beta;</td><td>BD</td><td>D21-1351</td><td>Mass: 158</td></tr><tr><td>MCP1</td><td>BD</td><td>5D3-F7</td><td>Mass: 170</td></tr><tr><td>IFN&amp;alpha;</td><td>Miltenyi&amp;nbsp;</td><td>LT27:295</td><td>Mass: 175</td></tr><tr><td>IFN&amp;gamma;</td><td>Biolegend</td><td>4S.B3</td><td>Mass: 165</td></tr><tr><td>PTEN</td><td>BD</td><td>A2B1</td><td>Mass: 159</td></tr><tr><td>TNF&amp;alpha;</td><td>Biolegend</td><td>Mab11</td><td>Mass: 166</td></tr><tr><td>&lt;strong&gt;Note&lt;/strong&gt;: If the manufacturer is stated as Fluidigm, this antibody was purchased from Fluidigm with metal pre-conjugated. If the manufacturer is stated as other than Fluidigm, this antibody was self-conjugated using the MaxPar Multi-Metal Labeling&amp;nbsp; Kit (Fluidigm Cat: 201300) according to manufacturer protocol.&amp;nbsp;</td><td></td><td></td><td></td></tr></tbody></table>

single-cell analysis of immunophenotype and cytokine production in peripheral whole blood via mass cytometry

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies in an attempt to understand the precise mechanisms that lead to the breakdown of self-tolerance and subsequent autoimmunity. Approaches thus far have been based on the study of one specific aspect of the immune system (a single or few cell types or cytokines), and do not offer a global assessment of complex autoimmune disease. While patient sera-based studies have afforded important insights into autoimmunity, they do not provide the specific cellular source of the dysregulated cytokines detected. A comprehensive single-cell approach to evaluate cytokine production in multiple immune cell subsets, within the context of &#34;intrinsic&#34; patient-specific plasma circulating factors, is described here. This approach enables monitoring of the patient-specific immune phenotype (surface markers) and function (cytokines), either in its native &#34;intrinsic pathogenic&#34; disease state, or in the presence of therapeutic agents (in vivo or ex vivo).

Figure 1 demonstrates the workflow for the stimulation and processing of the peripheral blood samples, including allocation of blood sample aliquots, timing of the addition of stimulation agents, protein transport inhibitor cocktail, and incubation times until the red blood cell (RBC) lysis and fixation. The choice of stimulating agents will depend on the signaling and cytokine pathways that are targeted for assessment. For example, in the protocol described ...

Watch this Scientific Journal Video about Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry at JoVE.com

Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry

Here we describe a single-cell proteomic approach to evaluate immune phenotypic and functional (intracellular cytokine induction) alterations in peripheral whole blood samples, analyzed via mass cytometry.

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies ...

single-cell-analysis-immunophenotype-cytokine-production-peripheral

Research

JoVE Journal

Immunology and Infection

9.4K Views.  University of Colorado School of Medicine. Here we describe a single-cell proteomic approach to evaluate immune phenotypic and functional (intracellular cytokine induction) alterations in peripheral whole blood samples, analyzed via mass cytometry.

Video: Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

Bioengineering

Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many advantages for ease and standardization of immunological assessments, but detrimental effects of this process have been observed on some cell subsets, such as granulocytes, B cells, and dendritic cells 1-3. Assaying fresh leukocytes gives a more accurate picture of the in vivo state of the cells, but is often difficult to perform in the context of large clinical trials. Fresh cell assays are dependent upon volunteer commitments and timeframes and, if time-consuming, their application can be impractical due to the working hours required of laboratory personnel. In addition, when trials are conducted at multiple centers, laboratories with the resources and training necessary to perform the assays may not be located in sufficient proximity to clinical sites. To address these issues, we have developed an 11-color antibody staining panel that can be used with Trucount tubes (Becton Dickinson; San Jose, CA) to phenotype and enumerate the major leukocyte populations within the peripheral blood, yielding more robust cell-type specific information than assays such as a complete blood count (CBC) or assays with commercially-available panels designed for Trucount tubes that stain for only a few cell types. The staining procedure is simple, requires only 100 &mu;l of fresh whole blood, and takes approximately 45 minutes, making it feasible for standard blood-processing labs to perform. It is adapted from the BD Trucount tube technical data sheet (<a href="http://www.bdbiosciences.com/external_files/is/doc/tds/Package_Inserts_CE/live/web_enabled/23-3483-06_PI_Trucount tubes_CE_EN.pdf" target="_blank">version 8/2010</a>). The staining antibody cocktail can be prepared in advance in bulk at a central assay laboratory and shipped to the site processing labs. Stained tubes can be fixed and frozen for shipment to the central assay laboratory for multicolor flow cytometry analysis. The data generated from this staining panel can be used to track changes in leukocyte concentrations over time in relation to intervention and could easily be further developed to assess activation states of specific cell types of interest. In this report, we demonstrate the procedure used by blood-processing lab technicians to perform staining on fresh whole blood and the steps to analyze these stained samples at a central assay laboratory supporting a multicenter clinical trial. The video details the procedure as it is performed in the context of a clinical trial blood draw in the HIV Vaccine Trials Network (HVTN).

In this report, we demonstrate the staining and analysis steps of a phenotyping assay performed on fresh whole blood to enumerate major innate and adaptive leukocyte populations. We emphasize considerations for performing these procedures in the context of a multicenter clinical trial.

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many ...

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease and treatment. It has the potential to predict therapeutic efficacy and identify novel therapeutic targets. Whole blood staining utilizes unmanipulated blood, which minimizes artifacts that can occur during sample preparation. However, whole blood staining must also be done on freshly collected blood to ensure the integrity of the sample. Additionally, it is best to prepare antibody cocktails on the same day to avoid potential instability of tandem-dyes and prevent reagent interaction between brilliant violet dyes. Therefore, whole blood staining requires careful standardization to control for intra and inter-experimental variability.
Here, we report deployment of an automated liquid handler equipped with a two-dimensional (2D) barcode reader into a standard process of making antibody cocktails for flow cytometry. Antibodies were transferred into 2D barcoded tubes arranged in a 96 well format and their contents compiled in a database. The liquid handler could then locate the source antibody vials by referencing antibody names within the database. Our method eliminated tedious coordination for positioning of source antibody tubes. It provided versatility allowing the user to easily change any number of details in the antibody dispensing process such as specific antibody to use, volume, and destination by modifying the database without rewriting the scripting in the software method for each assay.
A proof of concept experiment achieved outstanding inter and intra- assay precision, demonstrated by replicate preparation of an 11-color, 17-antibody flow cytometry assay. These methodologies increased overall throughput for flow cytometry assays and facilitated daily preparation of the complex antibody cocktails required for the detailed phenotypic characterization of freshly collected anticoagulated peripheral blood.

Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. Setup of a classic multicolor panel requires high-end instruments, custom labeled antibodies, and careful study design to minimize spectral overlap. We developed a multiparametric analysis to identify major human immune populations (CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes) in peripheral blood by combining seven lineage markers using only two fluorochromes. Our strategy is based on the observation that lineage markers are constantly expressed in a unique combination by each cell population. Combining this information with a careful titration of the antibodies allows investigators to record five additional markers, expanding the optical limit of most flow cytometers. Head-to-head comparison demonstrated that the vast majority of immune cell populations in peripheral blood can be characterized with comparable accuracy between our method and the classic &#34;one fluorochrome-one marker approach&#34;, although the latter is still more precise for identifying populations such as NKT cells and &#947;&#948; T cells. Combining seven markers using two fluorochromes allows for the analysis of complex immune cell populations and clinical samples on affordable 6-10 fluorochrome flow cytometers, and even on 2-3 fluorochrome field instruments in areas with limited resources. High-end instruments can also benefit from this approach by using extra fluorochromes to accomplish deeper flow cytometry analysis. This approach is also very well suited for screening several cell populations in the case of clinical samples with limited number of cells.

Here, we present a flow cytometric protocol to identify CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes in human peripheral blood by using only two fluorochromes instead of seven. With this approach, five additional markers can be recorded on most flow cytometers.

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...

Optimized Whole Blood Immunoassays for Analyzing Fungal and Viral Immunity

Whole Blood Assay with Dual Co-Stimulation for Antigen-Specific Analysis of Host Immunity to Fungal and Viral Pathogens

Rapid and resource-efficient sample processing, high throughput, and high robustness are critical for effective scientific and clinical application of advanced antigen-specific immunoassays. Traditionally, such immunoassays, especially antigen-specific T-cell analysis by flow cytometry or enzyme-linked immunosorbent spot assays, often rely on the isolation of peripheral blood mononuclear cells. This process is time-consuming, subject to many pre-analytic confounders, and requires large blood volumes. Whole blood-based assays provide a facile alternative with increased pre-analytic robustness and lower blood volume requirements. Furthermore, whole blood-based assays allow for the preservation of inter-cellular interactions that are not captured by assays using isolated cell subsets. Recently, a refined whole blood immunoassay with dual anti-CD28 and anti-CD49d co-stimulation for comprehensive analysis of both antigen-specific T-cell functions and complex intercellular interactions in response to various fungal and viral antigens has been proposed. This protocol provides guidance for the preparation of stimulation tubes, blood stimulation, and downstream sample processing for flow cytometry, cytokine secretion assays, and transcriptional analyses. This includes a validated and functionally equivalent, previously unpublished, low-volume protocol (250 &#181;L) to make flow cytometric and cytokine-based T-cell monitoring more accessible for studies in pediatric patients or preclinical studies in small animals (e.g., mice). Altogether, these protocols provide a versatile toolbox for complex antigen-specific immune analysis in both clinical and translational research settings.

Author Spotlight: Advancing Immune Monitoring in Critical Care Patients Using Whole Blood Assays

Whole blood-based immunoassays provide a facile and resource-efficient tool to analyze antigen-specific immunity for diagnostic and research purposes. This article provides an optimized whole blood-based protocol with dual co-stimulation for comprehensive analysis of host immunity to fungal and viral pathogens, including a low-volume version for pediatric patients and small animals.

Rapid and resource-efficient sample processing, high throughput, and high robustness are critical for effective scientific and clinical application of ...

Sample Preparation for Mass Cytometry Analysis

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities for deep analysis well beyond what is possible with conventional fluorescence-based flow cytometry. As with any new technology, there are critical steps that help ensure the reliable generation of high-quality data. Presented here is an optimized protocol that incorporates multiple techniques for the processing of cell samples for mass cytometry analysis. The methods described here will help the user avoid common pitfalls and achieve consistent results by minimizing variability, which can lead to inaccurate data. To inform experimental design, the rationale behind optional or alternative steps in the protocol and their efficacy in uncovering new findings in the biology of the system being investigated is covered. Lastly, representative data is presented to illustrate expected results from the techniques presented here.

This article describes the collection and processing of samples for mass cytometry analysis.

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities ...

Biology

Mass Cytometry Analysis of Systemic and Local Immune Responses in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most common and deadliest forms of liver cancer worldwide. Despite advances in treatment, the prognosis for HCC patients remains poor due to the complex interplay of genetic, environmental, and immunological factors driving its progression. Understanding the immune landscape of HCC is crucial for developing effective therapies, particularly in the field of immunotherapy, which holds great promises for improving patient outcomes. This study employs mass cytometry (cytometry by time-of-flight [CyTOF]) technology to investigate both systemic and local immune responses in patients with HCC. By analyzing peripheral blood and tumor samples, the research aims to identify unique immune cell populations, and their functional states associated with HCC progression. The findings provide a comprehensive overview of the immune landscape in HCC, highlighting potential biomarkers and therapeutic targets. This approach offers valuable insights into the immune mechanisms underlying HCC and paves the way for the development of more effective immunotherapies for this malignancy.

This protocol outlines a comprehensive mass cytometry (cytometry by time-of-flight [CyTOF]) analysis method for evaluating both systemic and local immune responses in hepatocellular carcinoma (HCC). The approach aims to provide insights into the immune landscape of HCC, offering a deeper understanding of the tumor microenvironment and the associated immune mechanisms.

Hepatocellular carcinoma (HCC) is one of the most common and deadliest forms of liver cancer worldwide. Despite advances in treatment, the prognosis for ...

A Flow Cytometry Assay to Identify Immune Cell Subsets in Peripheral Blood Mononuclear Cells

Source: Torchia, M. L. G., et al. <a href="https://app.jove.com/v/58955">Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry.</a> J. Vis. Exp. (2019)
This video demonstrates a flow cytometry-based technique using two fluorochromes to identify CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, natural killer (NK) cells, and monocytes in human peripheral blood mononuclear cells (PBMCs). Upon incubating the cells with fluorochrome-conjugated antibodies against the surface markers, the cell subsets are identified using an optimal flow cytometric gating strategy.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
NOTE: This protocol has been tested ...