title: Cell-based flow cytometry assay to measure cytotoxic activity.
abstract: 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, LU₃₀/10(6) 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.
authors.affiliation: Vaccine and Gene Therapy Institute of Florida.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Noto
authors.affiliation: Vaccine and Gene Therapy Institute of Florida.
authors.forename: Pearline
authors.initials: P
authors.lastname: Ngauv
authors.affiliation: Vaccine and Gene Therapy Institute of Florida; ltrautmann@vgtifl.org.
authors.forename: Lydie
authors.initials: L
authors.lastname: Trautmann
mesh_terms: D015496:CD4-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology*; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology*; D002450:Cell Communication / Q000276:immunology; D003602:Cytotoxicity, Immunologic; D018984:Epitopes, T-Lymphocyte / Q000276:immunology*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D016176:T-Lymphocyte Subsets / Q000166:cytology / Q000276:immunology
chemical_list: D018984:Epitopes, T-Lymphocyte