Intracellular Flow Cytometry for T Cell Analysis

Intracellular flow cytometry analysis was performed as previously described (22 (link)). Briefly, 5 × 10⁶ splenocytes were plated in each well of a 24-well plate and incubated with either CMFO (10 µg/mL) or PPD (10 µg/mL) in the presence of 1 µg/mL anti-CD28/CD49d (eBioscience CA, USA). RPMI1640 medium and cell stimulation cocktail (eBioscience, CA, USA) were used as negative and monitoring controls, respectively. Cells were stained with surface markers, including anti-CD4 PE Cy7, anti-CD8a PE, anti-CD44 APC-eFluor^® 780, anti-CD62L FITC mAbs, and intracellular markers anti-IFN-γPerCP-Cy5.5 and anti-IL-2 APC mAbs (all from eBioscience, CA, USA). The stained cells were analyzed by an LSRII multicolor flow cytometer (BD Biosciences, CA, USA). The absolute number of CMFO-specific CD4⁺ or CD8⁺ IFN-γ positive T_EM (effector memory T cells, CD62L^loCD44^hi) and CD4⁺ or CD8⁺ IL-2 positive T_CM (central memory T cells, CD62L^hiCD44^hi) cells were analyzed with FlowJo software (Tree Star Inc., OH, USA). The results are represented as mean ± SD per group (n = 6).