Multiparametric Flow Cytometry Analysis

Pooled splenocytes (2×10⁵ cells/group, n = 3-4 / group) obtained from naive control, MPTP and calpeptin treated mice were stained with CD8-FITC (BD Biosciences, San Jose, CA) and CD4-PerCP (BD Biosciences, San Jose, CA) as described [20 (link)]. Flow cytometric two-parameter dot plots and quadrant statistics were generated using FACScan and CellQuest software (BD Biosciences, Mountain view, CA). For intracellular staining, cells were fixed and permeabilized using Fix and Perm reagents (BD Biosciences, San Jose, CA), and then incubated with specific antibodies [75 (link)]. Briefly, 5×10⁵ cells per group were re-suspended in 0.5 mL of Fixation/Permeabilization Buffer (BD) and incubated at 2-8° C for 30 minutes. After washing, the cell pellet was re-suspended in Permeabilization/Wash Buffer and stained with Foxp3-APC (BD Biosciences, San Jose, CA). Cells were then analyzed on FACScan using CellQuest software as described above.