Cell-baserade flödescytometri analys för att mäta cytotoxisk aktivitet

1Vaccine and Gene Therapy Institute of Florida
Immunology and Infection

Your institution must subscribe to JoVE's Immunology and Infection section to access this content.

Fill out the form below to receive a free trial or learn more about access:


Enter your email below to get your free 10 minute trial to JoVE!

We use/store this info to ensure you have proper access and that your account is secure. We may use this info to send you notifications about your account, your institutional access, and/or other related products. To learn more about our GDPR policies click here.

If you want more info regarding data storage, please contact gdpr@jove.com.


Cite this Article

Copy Citation | Download Citations

Noto, A., Ngauv, P., Trautmann, L. Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity. J. Vis. Exp. (82), e51105, doi:10.3791/51105 (2013).

Please note that all translations are automatically generated.

Click here for the english version. For other languages click here.


Cytolytisk aktivitet av CD8 + T-celler sällan utvärderas. Vi beskriver här en ny cellbaserad analys för att mäta förmågan hos antigenspecifika CD8 + T-celler till att döda CD4 + T-celler laddade med deras besläktade peptiden. Target CD4 + T-celler är uppdelade i två populationer, som är märkta med två olika koncentrationer av CFSE. En befolkning pulsas med peptiden av intresse (CFSE-låg) medan den andra förblir oför pulsad (CFSE-hög). Pulsed och un-pulserade CD4 +-T-celler blandas vid ett lika förhållande och inkuberades med ett ökande antal av renade CD8 +-T-celler. Den specifika dödandet av autologa mål-CD4 +-T-celler analyserades med flödescytometri efter samodling med CD8 +-T-celler som innehåller den antigenspecifika effektor-CD8 + T-celler som detekteras av peptid / MHCI tetramer-färgning. Den specifika lys av mål-CD4 +-T-celler mäts vid olika effektor kontra målgrupp nyckeltal, möjliggör beräkning av lytiska enheter, LU 30/10 6 celler. Denna enkla och straightforward analysen möjliggör en noggrann mätning av den inneboende kapaciteten hos CD8 + T-celler till att döda mål-CD4 +-T-celler.


Name Company Catalog Number Comments
EasySep Human CD4+ T Cell Enrichment Kit Stem Cell 19052
EasySep Human CD8+ T Cell Enrichment Kit Stem Cell 19053
CellTrace CFSE Cell Proliferation Kit - For Flow Cytometry Invitrogen C34554
Far Red LIVE/DEAD fixable dead cell Invitrogen L10120
Alexa Fluor 700 Mouse Anti-Human CD3 BD 557943
Brilliant Violet 650 Anti-Human CD4 Antibody Biolegend 317435
PerCP anti-human CD8 Antibody Biolegend 344707
RPMI 1640 Medium 1x VWR 10-040-CV
Penicillin-Streptomycin 100x Solution Invitrogen 15070063
Fetal Bovine Serum - Gold, Heat Inactivated PAA A15-252
96-well plates; Treated; U-bottom; with lid BD Falcon 353077
25 ml sterile polystyrene, individually wrapped reservoirs USA Scientific 2321-2230
96-well U-bottom, sterile 2 ml deep well plates Thermo Scientific 275743
Human IL-2 (v126), research grade Miltenyi Biotec 130-093-096
RoboSep Buffer, separation buffer Stem cell 20104
15 ml Polypropylene Conical Tube BD Falcon 352097
14 ml round bottom tube, polypropylene BD Falcon 352059
The Big Easy EasySep Magnet Stem cell 18001
PBS 1x VWR 21-040-CV
96-well; V-bottom plate with lid Fisher 12-565-481
Formaldehyde solution, ACS reagent, 37 wt % in H2O Sigma-Aldrich 252549-500ML
1.5 ml screw cap microtube, conical Sarstedt 72.692.005



  1. Appay, V., Van Lier, R. A. W., Sallusto, F., Roederer, M. Cytometry. 73, 975-983 (2008).
  2. Betts, M. R., Harari, A. Phenotype and function of protective T cell immune responses in HIV. Curr. Opin. HIV AIDS. 3, 349-355 (2008).
  3. Roder, J. C., et al. A new immunodeficiency disorder in humans involving NK cells. Nature. 284, 553-555 (1980).
  4. Barber, D. L., Wherry, E. J., Ahmed, R. Cutting edge: rapid in vivo killing by memory CD8 T cells. J. Immunol. 171, 27-31 (2003).
  5. Byers, A. M., Kemball, C. C., Moser, J. M., Lukacher, A. E. Cutting edge: rapid in vivo CTL activity by polyoma virus-specific effector and memory CD8+ T cells. J. Immunol. 171, 17-21 (2003).
  6. Devevre, E., Romero, P., Mahnke, Y. D. LiveCount Assay: concomitant measurement of cytolytic activity and phenotypic characterisation of CD8(+) T-cells by flow cytometry. J. Immunol. Methods. 311, 31-46 (2006).
  7. Altman, J. D., et al. Phenotypic analysis of antigen-specific T lymphocytes. Science. 274, 94-96 (1996).
  8. Mbitikon-Kobo, F. M., Bonneville, M., Sekaly, R. P., Trautmann, L. Ex vivo measurement of the cytotoxic capacity of human primary antigen-specific CD8 T cells. J. Immunol. Methods. 375, 252-257 (2012).
  9. Bryant, J., Day, R., Whiteside, T. L., Herberman, R. B. Calculation of lytic units for the expression of cell-mediated cytotoxicity. J. Immunol. Methods. 146, 91-103 (1992).
  10. Varadarajan, N., et al. A high-throughput single-cell analysis of human CD8(+) T cell functions reveals discordance for cytokine secretion and cytolysis. J. Clin. Invest. 121, 4322-4331 (2011).
  11. Trautmann, L., et al. Profound metabolic, functional, and cytolytic differences characterize HIV-specific CD8 T cells in primary and chronic HIV infection. Blood. 120, 3466-3477 (2012).
  12. Hildemann, S. K., et al. High Efficiency of Antiviral CD4(+) Killer T Cells. PLoS One. 8, (2013).



    Post a Question / Comment / Request

    You must be signed in to post a comment. Please or create an account.

    Usage Statistics