Anti cd8 apc rpa t8

Splenocytes were stimulated for 6 h in complete RPMI medium at 37 °C in 5% CO₂ in the presence of 2 µg/ml peptide pool and 4 µg/ ml Brefeldin A (last 4 h). Unstimulated cells were used to assess nonspecific/background cytokine production. After 6 h, cells were harvested and stained for cell surface markers anti-CD4-PE (L200), anti-CD3-BV421 (SP34-2, both from BD Biosciences), anti-CD8-APC (RPA-T8), and Live/Dead Aqua (both from eBioscience). After washing, cells were fixed and permeabilized using 4% paraformaldehyde (PFA) and saponin buffer and then stained for cytokine using anti- IFN-γ-FITC (4S.B3, eBiosciences). Samples were acquired on a BD LSR II flow cytometer. The frequency of cells responding to the Mtb-specific peptides was quantified by determining the total number of cytokine⁺ cells and background values subtracted (as determined from the medium alone control) using FlowJo software (Tree Star). Lymphocytes, gated based on forward and side scatter, were gated for live CD3⁺ T cells. Live CD3⁺ T cells were then gated based on CD4 and CD8 expression. Then the IFN-γ⁺ cells were identified from these populations.