Multiparameter Flow Cytometry for Cellular Phenotyping

The following fluorophore conjugated monoclonal antibodies were used: CD3 (clone SP34-2), CD4 (clone L200), CD95 (clone DX2), Ki67 (clone B56), CD16 (clone 3G8), γδ TCR (clone B1), IFN-© (clone B27), TNFα (clone Mab11) (BD Biosciences), Perforin (clone Pf-344) (Mabtech), CD107a (clone eBioH4A3), CD28 (clone CD28.2); CD8 (clone 3B5) and Granzyme B (clone GB12; Life Technologies). Briefly, lymphocyte single cell suspensions were washed with PBS supplemented with 0.2% heat-inactivated human serum (Sigma), and incubated with different cocktails of fluorophore-labelled monoclonal antibodies during 20 minutes at room temperature [73 (link)], fixed and permeabilized using the FoxP3 permeabilization reagent (eBioscience). After 30 minutes incubation at 4°C, the cells were washed with FoxP3 washing buffer and intracellularly stained with Ki67 and GrzB for 20 minutes. The cells were washed and resuspended in PBS for flow cytometry analysis. For tetramer staining using samples from MamuA*01⁺ macaques, the CM9 tetramer was added to the samples 5 minutes prior to the addition of the antibody cocktail for surface staining [73 (link)]. The samples were acquired on a Fortessa or LSRII flow cytometer (BD Biosciences, San Jose, CA) and the data were analyzed using the FlowJo software platform (Tree Star, Inc., Ashland, OR).