We thank Dr. Dhelio Pereira and the members of Research Center for Tropical Medicine of Rond&#244;nia (CEPEM) for malaria patient enrollment and blood collection and Felicia Ho for helping with manuscript revision. The following reagent was obtained through BEI Resources, NIAID, NIH: Plasmodium yoelii subsp. yoelii, Strain 17XNL:PyGFP, MRA- 817, contributed by Ana Rodriguez. This research was supported by Lemann Brazil Research Fund, Conselho Nacional de Desenvolvimento Cienti&#769;fico e Tecnolo&#769;gico (CNPq) - 437851/2018-4, fellowships (CJ, GC, CG), and Funda&#231;&#227;o de Amparo do Estado de Minas Gerais (FAPEMIG) - APQ-00653-16, APQ-02962-18; Coordena&#231;&#227;o de Aperfei&#231;oamento de Pessoal de N&#237;vel Superior (CAPES) - fellowship (LL).

All procedures were conducted following the policies of the Oswaldo Cruz Foundation and the National Ethical Council (CAAE: 59902816.7.0000.5091). The human protocols were developed in collaboration with the clinical research group from the Research Center for Tropical Medicine of Rond&#244;nia (CEPEM), which was in charge of enrolling patients in the study. Informed consent was obtained from all the patients.
For the animal study, procedures were performed following the principles of conduct of the Brazilian Practice Guide for the Care and Use of Animals for Scientific and Didactic Purposes of the National Council for the Control of Animal Experimentation (CONCEA). The protocols were approved by the Fiocruz Animal Experimentation Council (CEUA protocol LW15/20-2).
1. Collection of human blood samples and PBMC isolation
<ol>
	<li>Collect blood of Plasmodium-infected patients in a 10 mL evacuated blood collection tube with sodium heparin. As malaria patients display lymphopenia, there is a range of 5-9 x 106 peripheral blood mononuclear cells (PBMCs) in a 10 mL blood sample. Since CD8+ T cells or &#947;&#948; T cells make up ~10% of PBMCs, preferably collect 50-100 mL of blood/patient. 
	NOTE: A minimum of 1 x 105 effector cells/ condition should be considered.</li>
	<li>Calculate the percentage of iRBCs by blood smear as described below.
	<ol>
		<li>Add 5 &#956;L of total blood to a clear slide and prepare a blood smear. Perform Romanowsky-type panoptic fast stain or May-Gu&#776;nwald-Giemsa stain. Here, the panoptic fast stain kit is used, which is composed of three reagents: reagent A (fixation), reagent B (cytoplasmic stain), and reagent C (nuclear and cytoplasmic differential stain).</li>
		<li>Slowly dip the slide in solution A 10x, then 4x in solution B, and finally 10x in solution C. Drain out any excess reagent from the slides between solutions. After dipping in solution C, rinse the slide in running tap water and let it dry.</li>
		<li>Under an upright light microscope with a 100x oil immersion objective, count 1000 RBCs in sequential squares and calculate the percentage of parasitemia using the following equation: 
		<img alt="Equation 1" src="/files/ftp_upload/63987/63987eq01.jpg" style="margin: auto;" /></li>
	</ol>
	</li>
	<li>Dilute 15 mL of blood in a 1:1 proportion in sterile phosphate-buffered saline (PBS).</li>
	<li>Add 15 mL of lymphocyte separation medium (density of 1.077g/mL) to a 50 mL tube. Carefully layer the 30 mL of diluted blood sample onto the centrifugation medium solution. When layering the sample, do not let the blood sample and lymphocyte separation medium mix.</li>
	<li>Centrifuge the tubes at 400 x g for 40 min at 22 &#176;C, with low acceleration and no break setting.</li>
	<li>Draw off the upper layer containing plasma using a sterile pipette, leaving the mononuclear cell layer undisturbed. Transfer the layer of mononuclear cells (PBMCs) to a sterile tube using a sterile pipette.</li>
	<li>Do not discard the tube containing the blood pellet as it will be used later for infected RBC isolation. From this step, be sure to keep the RBCs at room temperature (RT). Never let them cool down.</li>
	<li>Wash the cells twice by adding PBS and centrifuge at 350 x g for 10 min, with break setting. Resuspend the cell pellet in 5 mL of RPMI medium supplemented with penicillin/streptomycin and 10% fetal bovine serum (FBS; complete medium).</li>
	<li>Count the PBMCs in the presence of trypan blue solution to check cell viability, using a hemacytometer (Neubauer chamber) or automated cell counter. Do not count the blue-stained cells as these represent dying cells, which absorb trypan blue.</li>
	<li>Adjust the cell concentration to 107 cells/mL using complete medium. Purify the desired cytotoxic lymphocyte populations (CD8+ T cells, NK cells, &#947;&#948; T cells) using magnetic beads isolation as per reagent manufacturer protocol.</li>
</ol>
2. Human RBC isolation
NOTE: For human-infected RBC isolation, it is recommended to start with blood samples that have a minimum of 2% parasitemia, preferentially with more in the trophozoite/early schizont parasite stage.
<ol>
	<li>Prepare the PERCOLL (from hereafter referred to as density gradient separation medium) at the recommended concentration as described below.
	<ol>
		<li>Add 90 mL of 100% density gradient separation medium and 10 mL of 10x PBS to obtain 90% isotonic density gradient separation medium.</li>
		<li>For P. vivax-infected reticulocyte separation, prepare45% density gradient separation media. Add 50 mL of 90% isotonic density gradient separation medium and 50 mL of 1x PBS to obtain 45% density gradient separation medium.</li>
		<li>For P. falciparum-infected erythrocyte separation, prepare 65% density gradient separation media. Add 72 mL of 90% isotonic density gradient separation medium and 28 mL of 1x PBS to obtain 65% density gradient separation medium.</li>
		<li>For uninfected reticulocyte separation, prepare 70% density gradient separation media. Add 78 mL of 90% isotonic density gradient separation medium and 22 mL of 1x PBS to obtain 70% density gradient separation medium.</li>
	</ol>
	</li>
	<li>Warm the density gradient separation medium to 37 &#176;C in a water bath.</li>
	<li>After removal of the PBMC layer, carefully remove as much of the top layer of centrifugation medium as possible without touching the cells. With a glass Pasteur pipette, carefully remove the upper neutrophil layer without disturbing the RBC pellet and estimate pellet volume. Add 4x the RBC pellet volume of RT complete medium and resuspend.</li>
	<li>In a second 50 mL conical tube, add 5x the pellet volume of 45%, 65%, or 70% density gradient separation medium depending on the cell type to isolate. Layer the RBC suspension on top of the density gradient separation medium layer carefully.</li>
	<li>Spin for 15 min at 850 x g, with low acceleration and no break setting. Collect the cloudy red/brown layer lying in between the supernatant and density gradient separation medium with a 5 mL pipette. Transfer to a sterile 15 mL tube.</li>
	<li>Wash the cells by adding RT complete medium up to 15 mL and spin down for 10 min at 860 x g. Repeat the washing by adding 10 mL of RT complete medium and spin down. Discard the supernatant and prepare a blood smear from the pellet to check for iRBC enrichment, following step 1.2.</li>
	<li>Resuspend the pellet in 1 mL of RT complete medium and let sit at room temperature while counting the cells. Add 10 &#956;L of cell suspension in a hemocytometer (Neubauer chamber) and count the RBCs in the central area subdivided into 25 medium squares. Adjust cell concentration to 1 x 107 iRBCs/mL in RT complete media.</li>
</ol>
3. Experimental malaria infection in mouse
<ol>
	<li>Thaw an aliquot of cryopreserved P. yoelii 17XNL:PyGFP (MRA-817), a GFP-expressing strain obtained from MR4/ATCC, and inject 100 &#956;L intraperitoneally (i.p.) in an 8-week-old female C57BL/6 mouse.</li>
	<li>Follow the parasitemia burden every 3 days by tail vein lancing blood collection.
	<ol>
		<li>Puncture the vessel with the needle bevel up, entering the vein at a shallow angle beginning at the distal end of the tail.</li>
		<li>Collect the blood sample with a pipette or capillary tube up to 5 or 10 mL, then apply manual pressure to stop the bleeding.</li>
		<li>Prepare a blood smear (as described in step 1.2) until parasitemia reaches 10%-15% of infected RBCs.</li>
	</ol>
	</li>
	<li>Collect 10 &#956;L of blood by tail vein lancing and dilute to 100 &#956;L of PBS to i.p. inject into the second donor mouse. 
	NOTE: Cryopreserved parasites should be thawed and passed in mice twice before being used for experimental infections.</li>
	<li>When the second passage reaches 15% of parasitemia, collect five drops of blood by the tail clipping method and dilute in 1 mL of PBS. Prepare an infection solution by adjusting the concentration to 1 x 106 iRBCs/mL in sterile PBS and inject i.p. 100 &#956;L (1 x 105 iRBCs) of the solution into each mouse required for the experiment.</li>
	<li>Monitor parasitemia every 2-3 days until it reaches ~30% iRBCs, which occurs approximately 12 days post-infection. When the mice reach the desired parasitemia, collect blood by cardiac puncture as described below.
	<ol>
		<li>Aspirate 100 &#181;L of heparin solution (30 U/mL) into a 1 mL syringe with a 26 G needle.</li>
		<li>Anesthetize the mouse by inhalation with 5% isoflurane and confirm the absence of reflexes. Place the mouse on its side and perpendicularly insert the needle just below the elbow, through the ribs, and into the heart. Pull out the syringe plunger slowly and rotate the needle until 0.5-1 mL of blood is obtained.</li>
		<li>Perform humane euthanasia by cervical dislocation under anesthesia. Aseptically clean the left side of the mouse with 70% ethanol. With surgical scissors, make a cut on the left side of the mouse passing through the skin and peritoneum. Locate the spleen and remove it.</li>
		<li>Place the mouse spleen in a Petri dish with 5 mL of complete medium to purify the desired effector cell population (e.g., CD8+ T cells).</li>
	</ol>
	</li>
</ol>
4. Obtaining fresh whole mouse splenocytes
<ol>
	<li>In the Petri dish, carefully cut the spleen into small pieces using scissors or a razor.</li>
	<li>Place a 100 &#181;M cell strainer over a 50 mL conical tube and transfer the excised spleen into the cell strainer with a pipette. Mash the spleen through the strainer with a syringe plunger.</li>
	<li>Wash the cells through the strainer with 10 mL of complete media. Centrifuge the cells at 300 x g for 10 min at 4 &#176;C, then discard the supernatant.</li>
	<li>Resuspend the cell pellet in 2 mL of cold 1x RBC lysis buffer. Incubate the suspension for 5 min on ice.</li>
	<li>Wash the cell suspension with 10 mL of 4 &#176;C complete media. Repeat the wash step three times and remove any cell clots between washes for spleens from Plasmodium-infected mice.</li>
	<li>Centrifuge the cells at 400 x g for 5 min at 4 &#176;C, then discard the supernatant. 
	NOTE: Plasmodium-infected spleens are enlarged and filled with phagocytic cells containing degraded hemoglobin and hemozoin.</li>
	<li>To avoid any issues during the magnetic bead isolation of effector cells, use the following steps to remove hemozoin-enriched phagocytic cells and hemozoin. Resuspend the splenic cells with MACS buffer and adjust concentration to 1 x 108 cells/mL. Place an LS or LD column in the magnetic field. Prepare the column by rinsing with 3 mL of MACS buffer.</li>
	<li>Apply cell suspension onto the column. Wash the column three times with 3 mL of MACS buffer. Collect flow-through containing the splenic cells.</li>
	<li>Count the splenocytes in a trypan blue solution and check cell viability in a hemacytometer (Neubauer chamber) or automated cell counter. Adjust cell concentration to 1 x 107 cells/mL in RT complete media.</li>
</ol>
5. Cytotoxic effector cell purification (CD8a+ T cell negative selection)
NOTE: There are many positive and negative selection reagents that can purify cytotoxic effector cells (CD8+ T, &#947;&#948; T, NK, iNKT, MAIT cells). In this protocol, we use a negative selection of splenic CD8a+ T cells and follow manufacturer instructions.
<ol>
	<li>Centrifuge all splenocytes at 400 x g for 10 min at 4 &#176;C and discard the supernatant. Resuspend the cell pellet in 40 &#181;L of MACS buffer.</li>
	<li>Add 10 &#181;L of a biotin-antibody cocktail. Mix well and incubate for 5 min on ice.</li>
	<li>Add 30 &#181;L of MACS buffer. Add 20 &#181;L of anti-biotin micro-beads. Mix well and incubate for 10 min on ice.</li>
	<li>Add 400 &#181;L of MACS buffer and proceed to magnetic cell separation. Place the MACS LS column in the magnetic field support. Prepare the column by rinsing with 3 mL of MACS buffer.</li>
	<li>Apply cell suspension onto the column. Wash the column three times with 3 mL of MACS buffer. Collect the flow-through containing all unlabeled cells, which are the enriched CD8a+ T cells.</li>
	<li>Count the CD8a+ T cells in a trypan blue solution and check cell viability in a hemacytometer (Neubauer chamber) or automated cell counter. Adjust the cell concentration to 1 x 107 cells/mL in RT complete media.</li>
</ol>
6. P. yoelii infected RBC isolation
<ol>
	<li>Centrifuge the collected blood in a 1.5 mL tube at 850 x g for 3 min. Discard the serum and resuspend the blood in 1 mL of 1x RPMI without FBS.</li>
	<li>Place the LS column in the magnetic field support and rinse with 3 mL of 1x RPMI. Pass the RBC suspension through the column. To isolate more iRBCs, reapply the flowthrough (3 mL of RPMI and 1 mL of diluted blood) into the column.</li>
	<li>Wash twice with 5 mL of 1x RPMI. Perform washing steps by adding buffer aliquots as soon as the column reservoir is empty. Do not let any columns dry up.</li>
	<li>Add 5 mL of RPMI, remove the column, and purge the iRBCs into a new 15 mL tube. Count the iRBCs and adjust the concentration to 1 x 107 RBCs/mL.</li>
</ol>
7. CFSE labeling of RBCs and preparation for flow cytometry
NOTE: Start the RBC labeling protocol with twice the number of cells that will be used for the experiment, since ~50% of the cells are typically lost in the washing steps following the CFSE labeling step. To establish the autofluorescence of the RBCs/iRBCs, include a control sample of unlabeled cells.
<ol>
	<li>Dilute CFSE to a final concentration of 10 mM in 1x RPMI without FBS. Wash the cells once with 1x RPMI without FBS in a 15 mL tube.</li>
	<li>Resuspend the RBCs in 500 &#956;L of 1x RPMI without FBS and add 500 &#956;L of the diluted CFSE. Incubate for 8 min at RT, protected from light.</li>
	<li>Wash three times by adding 14 mL of complete medium (10% FBS RPMI), followed by centrifugation at 850 x g for 10 min. Resuspend the cells in complete medium to a concentration of 1-5 x 106/ mL. Incubate for 1 h at RT.</li>
</ol>
8. Cytotoxic lymphocyte/RBC coculture and preparation for flow cytometry
NOTE: If the involvement of specific receptors or molecules will be measured, incubate the specific blocking and isotype control antibodies (10 mg/mL) with the effector cells for 30 min before coculture.
<ol>
	<li>Plate the cells in a 96-well round-bottom plate. Add the purified lymphocytes and CFSE-labeled iRBCs in the desired effector-target cell ratio to a final volume of 200 &#956;L and homogenize. Prepare each condition in triplicate. 
	NOTE: We suggest adjusting the iRBC concentration to 1-5 x 104 cells/mL and choosing the ratio based on the purified cell number (e.g., 0.5:1, 2.5:1, and 5:1).</li>
	<li>Include the spontaneous lysis control, which is the target iRBCs without the effector, to evaluate any spontaneous lysis, as this will be used to represent a 100% cell viability condition.</li>
	<li>Spin down the plate for 1 min at 360 x g to maximize cell contact. Incubate at 37 &#176;C and 5% CO2&#160;for 4 h. 
	NOTE: Hypoxia conditions (low O2), systematically used for P. falciparum culture, should not be used since the effector cells do not survive in this environment. In contrast, Plasmodium parasites are viable in ambient air in 5% CO2&#160;for up to 12 h.</li>
	<li>Spin for 5 min at 850 x g and flip the plate to remove the supernatant. 
	NOTE: If desired, the supernatant can be used to measure any soluble factors released in the coculture.</li>
	<li>Label the RBCs with anti-mouse Ter119 1:200 (or anti-human CD235 1:100 for human samples) and CD8+ T cells with anti-CD8 1:200 or anti-CD3 1:200 antibodies (anti-mouse or human) for 30 min at 4 &#176;C in 1x PBS containing 3% FBS (FACS buffer). 
	NOTE: The antibody fluorophore should be selected based on the cell tracer/cell proliferation reagent (e.g., CFSE-labelled iRBC, APC-Cy7 anti-Ter119, and PerCP-Cy5.5 anti-CD8a).</li>
	<li>Wash the cells with FACS buffer and spin down for 5 min at 850 x g. Transfer the samples to FACS tubes and add 30 &#956;L of counting beads &#65279;to individual tubes. Homogenize the count beads by vortexing for 30 s.</li>
</ol>
9. Flow cytometry
<ol>
	<li>Analyze samples using a 405/488/561/640 laser instrument.</li>
	<li>For human cells labeled with CFSE (FITC), PE anti-human &#947;&#948; TCR, and PE-Cy7 anti-human CD235a antibody, use 530/30 (FITC), 575/25 (PE), and 780/60 (PE-Cy7) filters in a three-laser (Blue, Red, Yellow-Green) configuration cytometer.</li>
	<li>For mouse cells labeled with CFSE (FITC), PerCP-Cy5.5 anti-mouse CD8a, and APC-Cy7 anti-mouse Ter119, use 530/30 (FITC), 695/40 (PerCP-Cy5.5), and 780/60 (APC-Cy7) filters in the three-laser cytometer setup.</li>
	<li>Choose the counting beads population as the stopping gate to acquire a minimum of 20,000 events in the stop gate, which should be identical for all conditions. Perform analysis and compensation using appropriate flow cytometry acquisition/analysis software.</li>
	<li>Set the gating strategy as described below.
	<ol>
		<li>Select single cells (singlets), excluding debris using FSC peak height (H) to area (A) ratio. Select RBCs and exclude the lymphocytes from analysis based on fluorescence of the RBC marker (Ter119 or CD235a) and the lymphocyte-specific antibody (CD8a).</li>
		<li>In the previous RBC gating, select viable RBCs based on CFSE positive&#160;staining. Analyze the data with flow cytometer data analysis software.</li>
	</ol>
	</li>
</ol>
10. Calculation and statistics
<ol>
	<li>To calculate the percentage of iRBC lysis, follow the gating strategy described in step 9. The percentage of viable RBCs is the frequency of CFSE-positive cells inside the RBC gate based on Ter119 (mouse) or CD235a (human) positive fluorescence.</li>
	<li>Use the spontaneous lysis control, RBCs without effector cells, condition as a control to estimate RBC spontaneous rupture. This condition will be considered RBC lysis (100% viability). Use the following formula to calculate the percentage of RBC lysis in each tested condition: 
	<img alt="Equation 2" src="/files/ftp_upload/63987/63987eq02v4.jpg" style="margin: auto;" /> 
	NOTE: During the culture incubation, some infected RBCs may spontaneously lyse or be ruptured by the parasite. Use the CFSE-positive RBC frequency of the spontaneous lysis condition, which contains no effector cells, as the baseline for lymphocyte cytotoxicity.</li>
	<li>Calculate the average of triplicates for each condition and determine statistical significance using two-way ANOVA at multiple comparisons.</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+Plasmodium+falciparum+growth+and+development+by+UV+flow+cytometry+using+an+optimized+Hoechst-thiazole+orange+staining+strategy.">Monitoring Plasmodium falciparum growth and development by UV flow cytometry using an optimized Hoechst-thiazole orange staining strategy.</a> Cytometry Part A:The Journal of the International Society for Analytical Cytology. 73 (6), 546-554 (2008).</li><li>Pattanapanyasat, K., 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=Culture+of+malaria+parasites+in+two+different+red+blood+cell+populations+using+biotin+and+flow+cytometry.">Culture of malaria parasites in two different red blood cell populations using biotin and flow cytometry.</a> Cytometry:The Journal of the International Society for Analytical Cytology. 25 (3), 287-294 (1996).</li><li>Bianco, A. E., Battye, F. L., Brown, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodium+falciparum:+rapid+quantification+of+parasitemia+in+fixed+malaria+cultures+by+flow+cytometry.">Plasmodium falciparum: rapid quantification of parasitemia in fixed malaria cultures by flow cytometry.</a> Experimental Parasitology. 62 (2), 275-282 (1986).</li><li>Jacobberger, J. W., Horan, P. K., Hare, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+malaria+parasite-infected+blood+by+flow+cytometry.">Analysis of malaria parasite-infected blood by flow cytometry.</a> Cytometry:The Journal of the International Society for Analytical Cytology. 4 (3), 228-237 (1983).</li><li>Bei, A. K., 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+flow+cytometry-based+assay+for+measuring+invasion+of+red+blood+cells+by+Plasmodium+falciparum.">A flow cytometry-based assay for measuring invasion of red blood cells by Plasmodium falciparum.</a> American Journal of Hematology. 85 (4), 234 (2010).</li><li>Montes, M., Jaensson, E. A., Orozco, A. F., Lewis, D. E., Corry, D. 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+general+method+for+bead-enhanced+quantitation+by+flow+cytometry.">A general method for bead-enhanced quantitation by flow cytometry.</a> Journal of Immunological Methods. 317 (1-2), 45-55 (2006).</li></ol>

The authors declare no competing interests.

Here we describe an in vitro assay to measure Plasmodium-infected red blood cell killing by cytotoxic lymphocytes. This assay &#65279;can help elucidate the mechanisms of cellular protective immunity to the malaria parasite&#39;s erythrocytic stage. The major advantage of this methodology is that it provides a quantitative assay of the cell-mediated killing of iRBCs that can be used to address many questions about how immune cells interact with different Plasmodium spp.
<p class="jove_conte...

Malaria remains a global health crisis, with more than 240 million cases and 627,000 malaria-related deaths reported in 20201. There are currently five parasitic species that can cause malaria in humans, out of which Plasmodium falciparum and Plasmodium vivax are the two most prevalent species. During Plasmodium infection,&#160;the liver or pre-erythrocytic stage is asymptomatic, and symptoms only occur during the parasite&#39;s asexual cycle in the erythrocytic stage. At this infection stage, thousands of merozoites derived from the liver stage are released into the bloodstream and infect red blood cells (RBCs). In the RBC, the parasites differentiate into trophozoites and schizonts by schizogony, until schizonts rupture the erythrocyte, releasing newly formed merozoites, repeating this blood cycle. &#65279;Repeated cycles of invasion, replication, and merozoite release result in an exponential growth of the parasite population and ultimately trigger disease symptoms2.
An important challenge in studying the immune response to malaria is that the Plasmodium spp. that infects humans does not infect laboratory animal models. Thus, Plasmodium-infected patient samples must be collected fresh and immediately processed and analyzed. However, in malaria-endemic areas, resources to access immunological and molecular mechanisms are limited. Due to these limitations, rodents are widely used as experimental models to investigate the immune response against Plasmodium infection. While P. berghei and P. chabaudi are often used as surrogates for P. falciparum infection, the non-lethal strain of P. yoelii&#160;17XNL also has many features in common with P. vivax, such as reticulocyte restricted infection3,4. The development of Plasmodium in vitro assays, that can be used for human or animal model-derived samples, is valuable in gaining a better understanding of the pathogenesis of malaria and comparing the immunological response elicited by different species of the parasite.
Protective antimalarial immunity is not completely understood either at the pre-erythrocytic stage nor the blood stage. &#65279;It is known that exposure to repeated infections results in partial acquired immunity, but sterile immunity is rarely developed5. &#65279;For decades, anti-Plasmodium protective immunity was mainly associated with the induction of neutralizing or opsonizing antibodies that prevent parasite invasion of host cells or lead to phagocytosis by antigen-presenting cells, respectively6. As a result, most efforts to produce antimalarial vaccines thus far have relied on inducing protective and long-lasting antibodies7,8. However, the sterile protection induced by vaccination with an attenuated sporozoite directly correlates with the activation and expansion of cytotoxic T lymphocytes8,9.
Recently, some studies of freshly isolated patient samples and in vitro cultures have demonstrated that innate or adaptative cytotoxic immune cells like CD8+ T10, &#947;&#948; T11, and NK cells12&#160;can directly eliminate Plasmodium-infected RBCs and its intracellular parasite in an effector:target ratio manner. These seminal findings defined an entirely new immune effector mechanism in the context of malaria. To dissect this novel antimalarial immunity, it is essential to explore cytotoxic effector mechanisms of killer cells against infected-RBCs (iRBCs) in natural infection or vaccination.
Here we present an in vitro assay that measures the cytotoxic activity of lymphocytes against malaria in the blood stage. This assay can then help elucidate the mechanisms of the cellular immune response against the Plasmodium erythrocyte stage. The target cells, iRBCs, are labeled with carboxyfluorescein succinimidyl ester (CFSE) to evaluate cell viability, and are then cocultured with effector cells like cytotoxic lymphocytes (CTL). This coculture is then evaluated by flow cytometry, using fluorescent markers for specific cell types. Finally, the percentage of iRBC lysis by CTL is calculated by dividing the experimental condition by the spontaneous rupture of RBCs and spontaneous lysis control, which occurs during incubation without the effector cell. Overall, this killing assay methodology can contribute to a better understanding of cell-mediated malaria immunity.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 &#956;M cell strainer</td>
			<td>Corning</td>
			<td>431752</td>
			<td></td>
		</tr>
		<tr>
			<td>96 Well Round (U) Bottom Plate&#160;</td>
			<td>Thermo Scientific</td>
			<td>12-565-65</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human CD235a (Glycophorin A) Antibody</td>
			<td>Biolegend</td>
			<td>349114</td>
			<td>Used - APC anti-human CD235, dilution 1:100</td>
		</tr>
		<tr>
			<td>Anti-human CD3 Antibody</td>
			<td>Biolegend</td>
			<td>317314</td>
			<td>Used - PB anti-human CD3, dilution 1:200</td>
		</tr>
		<tr>
			<td>Anti-human CD8 Antibody</td>
			<td>Biolegend</td>
			<td>344714</td>
			<td>Used - APC/Cy7 anti-human CD8, dilution 1:200</td>
		</tr>
		<tr>
			<td>Anti-human TCR V&#948;2 Antibody</td>
			<td>Biolegend</td>
			<td>331408</td>
			<td>Used - PE anti-human TCR V&#948;2, dilution 1:200</td>
		</tr>
		<tr>
			<td>Anti-mouse CD8a Antibody&#160;</td>
			<td>Biolegend</td>
			<td>100733</td>
			<td>Used- PerCP/Cyanine5.5 anti-mouse CD8a, dilution 1:200</td>
		</tr>
		<tr>
			<td>Anti-mouse TER-119/Erythroid Cells Antibody</td>
			<td>Biolegend</td>
			<td>116223</td>
			<td>Used - APC/Cyanine7 anti-mouse TER-119, dilution 1:200</td>
		</tr>
		<tr>
			<td>CellTrace CFSE Cell Proliferation Kit</td>
			<td>Invitrogen</td>
			<td>C34554</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, qualified</td>
			<td>Gibco</td>
			<td>26140079</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll-Paque Plus&#160;</td>
			<td>Cytiva</td>
			<td>17144003</td>
			<td>Lymphocyte Separation Medium (LSM)</td>
		</tr>
		<tr>
			<td>Heparin Sodium Injection, USP</td>
			<td>meithel pharma</td>
			<td>71228-400-003</td>
			<td>Used - 2000 USP units/2mL</td>
		</tr>
		<tr>
			<td>Isoflurane&#160;</td>
			<td>Piramal critical care&#160;</td>
			<td>66794-0013-25</td>
			<td></td>
		</tr>
		<tr>
			<td>LS MACS Column</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>LSRFortessa Cell Analyzer</td>
			<td>BD Bioscience&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>Cytiva</td>
			<td>17089101</td>
			<td>Density Gradient Separation Medium (DGSM)</td>
		</tr>
		<tr>
			<td>QuadroMACS Separator</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-976</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium</td>
			<td>Gibco</td>
			<td>11875093</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate, powder,&#160; BioReagent</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>&#160;S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe With Sub-Q needle - 1mL, 26 gauge;&#160;</td>
			<td>BD</td>
			<td>14-829-10F</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacutainer Heparin Tube Glass Green 10 ml&#160;</td>
			<td>BD</td>
			<td>366480</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro assay of plasmodium-infected red blood cell killing by cytotoxic lymphocytes

Malaria is a major public health concern, presenting more than 200 million cases per year worldwide. Despite years of scientific efforts, protective immunity to malaria is still poorly understood, mainly due to methodological limitations of long-term Plasmodium culture, especially for Plasmodium vivax. Most studies have focused on adaptive immunity protection against malaria by antibodies, which play a key role in controlling malaria. However, the sterile protection induced by attenuated Plasmodium sporozoites vaccines is related to cellular response, mainly to cytotoxic T lymphocytes, such as CD8+ and gamma delta T cells (&#947;&#948; T). Hence, new methodologies must be developed to better comprehend the functions of the cellular immune response and thus support future therapy and vaccine development. To find a new strategy to analyze this cell-mediated immunity to Plasmodium blood-stage infection, our group established an in vitro assay that measures infected red blood cell (iRBC) killing by cytotoxic lymphocytes. This assay can be used to study cellular immune response mechanisms against different Plasmodium spp. in the blood stage. Innate and adaptative cytotoxic immune cells can directly eliminate iRBCs and the intracellular parasite in an effector:target mechanism. Target iRBCs are labeled to evaluate cell viability, and cocultured with effector cells (CD8+ T, &#947;&#948; T, NK cells, etc.). The lysis percentage is calculated based on tested conditions, compared to a spontaneous lysis control in a flow cytometry-based assay. Ultimately, this killing assay methodology is a major advance in understanding cell-mediated immunity to blood-stage malaria, helping uncover new potential therapeutic targets and accelerate the development of malaria vaccines.

Here, the applied methodology for isolation of CFSE-labeled Plasmodium-infected RBCs in a coculture assay with cytotoxic lymphocytes is described. First, we provide a schematic representation of how to perform the protocol, employing human samples infected with P. vivax (Figure 1). Then, an illustrated flowchart on how to proceed with the protocol in a malaria experimental model using a C57BL/6 mouse infected with P. yoelii (Figure 2)....

Watch this Scientific Journal Video about In Vitro Assay of Plasmodium-Infected Red Blood Cell Killing by Cytotoxic Lymphocytes at JoVE.com

In Vitro Assay of Plasmodium-Infected Red Blood Cell Killing by Cytotoxic Lymphocytes

Here we describe a new method to help elucidate the mechanisms of cellular immunity to Plasmodium during the blood stage of infection. This is an in vitro assay that measures infected red blood cell killing by cytotoxic lymphocytes.&#160;

In Vitro Assay of Plasmodium-Infected Red Blood Cell Killing by Cytotoxic Lymphocytes

Malaria is a major public health concern, presenting more than 200 million cases per year worldwide. Despite years of scientific efforts, protective ...

in-vitro-assay-plasmodium-infected-red-blood-cell-killing-cytotoxic

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JoVE Journal

Immunology and Infection

1.9K Views.  Fundação Oswaldo Cruz. Here we describe a new method to help elucidate the mechanisms of cellular immunity to Plasmodium during the blood stage of infection. This is an in vitro assay that measures infected red blood cell killing by cytotoxic lymphocytes. 

Video: In Vitro Assay of Plasmodium-Infected Red Blood Cell Killing by Cytotoxic Lymphocytes

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known phenotype.  Other bulk methods of purification include panning, complement depletion and magnetic bead separation.  However, FACS has several advantages over other available methods.  FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density.  In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker.  FACS allows the purification of individual cells based on size, granularity and fluorescence.  In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1).  Negative selection of unstained cells is also possible.  FACS purification requires a flow cytometer with sorting capacity and the appropriate software.  For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser.  The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells.  The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2).  The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb.  Sorting parameters can be adjusted depending on the requirement of purity and yield.  Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation. This labeling has classically been used to study proliferation and migration. In the method presented here, we have shortened the timeline after adoptive transfer to look at survival and killing of epitope specific CFSE labeled target cells4-6. The level of specific killing of a CD8 + T cell clone can indicate the quality of the response, as their quantity may be misleading. Specific CD8+ T cells can become functionally exhausted over time with a decline in cytokine production and killing7,8. Also, certain CD8 + T cell clones may not kill as well as others with differing TCR specificities9. For effective Cell Mediated Immunity (CMI), antigens must be identified that produce not only adequate numbers of responding T cells, but also functionally robust responding T cells. Here we assess the percent cell specific killing of two peptide specific T cell clones in BALB/c mice.

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Many infections elicit a strong CTL response, but occasionally, the quantity of responding cells does not correlate to control of the pathogen1. One measure of CTL quality is their ability to kill specifically2. CFSE labeling of target cells can be used to investigate this CTL response quality in vivo3,4.

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation.  ...

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as 
insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during 
the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the 
radionuclide Chromium 51 (51Cr) to label target cells. These targets are then exposed to effector cells and the release of 51Cr from target 
cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of 51Cr.
 As effector cells, we used an activated autoreactive clonal population of CD8+ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with 51Cr labeled target NIT cells for 16 hours, release of 51Cr was recorded to calculate specific lysis 
Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37&deg;C, with 5% CO2, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.
Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection.1-4 We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective.5-8 However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells. 
In an effort to better mimic the in vivo priming conditions of na&iuml;ve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus.9 At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15.10 At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2. 
From 50x106 CB mononuclear cells we are able to generate upwards of 150 x 106 virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation.11 These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.

Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly na&#238;ve T cells.

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell anemia 1-3. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens 4), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs 4, 5. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen ,glucose, and ions3. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers 6, 7, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane 8, 9 and mask RBC surface antigens10, 11.

The cell membrane modification of red blood cells (RBCs) with hyperbranched polyglycerol (HPG) is presented. Modified RBCs were characterized by aqueous two phase partitioning, osmotic fragility and complement mediated lysis. The camouflage of surface proteins and antigens was evaluated using the flow cytometry and Micro Typing System (MTS) blood phenotyping cards.

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell ...

Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

Phospholipid bilayers that constitute endo-lysosomal vesicles can pose a barrier to delivery of biologic drugs to intracellular targets. To overcome this barrier, a number of synthetic drug carriers have been engineered to actively disrupt the endosomal membrane and deliver cargo into the cytoplasm. Here, we describe the hemolysis assay, which can be used as rapid, high-throughput screen for the cytocompatibility and endosomolytic activity of intracellular drug delivery systems. 
In the hemolysis assay, human red blood cells and test materials are co-incubated in buffers at defined pHs that mimic extracellular, early endosomal, and late endo-lysosomal environments. Following a centrifugation step to pellet intact red blood cells, the amount of hemoglobin released into the medium is spectrophotometrically measured (405 nm for best dynamic range). The percent red blood cell disruption is then quantified relative to positive control samples lysed with a detergent. In this model system the erythrocyte membrane serves as a surrogate for the lipid bilayer membrane that enclose endo-lysosomal vesicles. The desired result is negligible hemolysis at physiologic pH (7.4) and robust hemolysis in the endo-lysosomal pH range from approximately pH 5-6.8.

Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

A hemolysis assay can be used as a rapid, high-throughput screen of drug delivery systems' cytocompatibility and endosomolytic activity for intracellular cargo delivery. The assay measures the disruption of erythrocyte membranes as a function of environmental pH.

Phospholipid bilayers that constitute endo-lysosomal vesicles can pose a barrier to delivery of biologic drugs to intracellular targets. To overcome this ...

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

Radial Mobility and Cytotoxic Function of Retroviral Replicating Vector Transduced, Non-adherent Alloresponsive T Lymphocytes

We report a novel adaptation of the Radial Monolayer Cell Migration assay, first reported to measure the radial migration of adherent tumor cells on extracellular matrix proteins, for measuring the motility of fluorescently-labeled, non-adherent human or murine effector immune cells. This technique employs a stainless steel manifold and 10-well Teflon slide to focally deposit non-adherent T cells into wells prepared with either confluent tumor cell monolayers or extracellular matrix proteins. Light and/or multi-channel fluorescence microscopy is used to track the movement and behavior of the effector cells over time. Fluorescent dyes and/or viral vectors that code for fluorescent transgenes are used to differentially label the cell types for imaging. This method is distinct from similar-type in vitro assays that track horizontal or vertical migration/invasion utilizing slide chambers, agar or transwell plates. The assay allows detailed imaging data to be collected with different cell types distinguished by specific fluorescent markers; even specific subpopulations of cells (i.e., transduced/nontransduced) can be monitored. Surface intensity fluorescence plots are generated using specific fluorescence channels that correspond to the migrating cell type. This allows for better visualization of the non-adherent immune cell mobility at specific times. It is possible to gather evidence of other effector cell functions, such as cytotoxicity or transfer of viral vectors from effector to target cells, as well. Thus, the method allows researchers to microscopically document cell-to-cell interactions of differentially-labeled, non-adherent with adherent cells of various types. Such information may be especially relevant in the assessment of biologically-manipulated or activated immune cell types, where visual proof of functionality is desired with tumor target cells before their use for cancer therapy.

We describe a protocol to monitor radial mobility of non-adherent immune cells in vitro using a cell sedimentation manifold/slide apparatus. Cell migration is tracked on monolayers of tumor cells or on extracellular matrix proteins. Examination by light and fluorescence microscopy allows for observation of cell mobility and cytotoxic functionality.

We report a novel adaptation of the Radial Monolayer Cell Migration assay, first reported to measure the radial migration of adherent tumor cells on ...

Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial and temporal resolution. FCS can quantify the molecular concentration and diffusion coefficient of fluorescently labeled molecules in the cell membrane. This technique has the ability to explore the molecular diffusion characteristics of molecules in the plasma membrane of immune cells in steady state (i.e., without processes affecting the result during the actual measurement time). FCS is suitable for studying the diffusion of proteins that are expressed at levels typical for most endogenous proteins. Here, a straightforward and robust method to determine the diffusion rate of cell membrane proteins on primary lymphocytes is demonstrated. An effective way to perform measurements on antibody-stained live cells and commonly occurring observations after acquisition are described. The recent advancements in the development of photo-stable fluorescent dyes can be utilized by conjugating the antibodies of interest to appropriate dyes that do not bleach extensively during the measurements. Additionally, this allows for the detection of slowly diffusing entities, which is a common feature of proteins expressed in cell membranes. The analysis procedure to extract molecular concentration and diffusion parameters from the generated autocorrelation curves is highlighted. In summary, a basic protocol for FCS measurements is provided; it can be followed by immunologists with an understanding of confocal microscopy but with no other previous experience of techniques for measuring dynamic parameters, such as molecular diffusion rates.

A method to measure protein diffusion in membranes of primary immune cells using fluorescence correlation spectroscopy (FCS) is described. In this paper, the use of antibodies for fluorescent labeling is illustrated.

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial ...