Pooled freshly isolated splenocytes (2×105cells/group, n=3-4 per group) obtained from naive control, MPTP, and calpeptin
treated mice were stained with CD8-FITC (BD Biosciences, San Jose, CA; #553031)
and CD4-PerCP (BD Biosciences, San Jose, CA: #553052) as previously described
(Samantaray et al., 2015 (link)). Other
antibodies used were: PE Granzyme B Monoclonal Antibody (eBioscience, Waltham,
MA, USA, #12-8898-82), APC anti-mouse Perforin antibody (BioLegend, San Diego,
CA, USA, #154304), and PE anti-mouse CD25 Antibody (BioLegend, San Diego, CA,
USA, #101904). Flow cytometric two-parameter dot plots and quadrant statistics
were generated using FACScan and CellQuest software (BD Biosciences, Mountain
view, CA, USA). For intracellular staining, cells were fixed and permeabilized
using Fix and Perm reagents (BD Biosciences, San Jose, CA) and incubated with
specific antibodies (God et al., 2015 ;
God et al., 2014 (link)). Briefly,
5×105 cells per group were re-suspended in 0.5 mL of
Fixation/Permeabilization Buffer and incubated at 2-8° C for 30 minutes.
After washing, the cell pellet was re-suspended in Permeabilization/Wash Buffer
and stained with CD4-PerCP, CD8-FITC, APC-perforin and PE-granzyme B. Cells were
then analyzed on FACScan using CellQuest software as described above.
treated mice were stained with CD8-FITC (BD Biosciences, San Jose, CA; #553031)
and CD4-PerCP (BD Biosciences, San Jose, CA: #553052) as previously described
(Samantaray et al., 2015 (link)). Other
antibodies used were: PE Granzyme B Monoclonal Antibody (eBioscience, Waltham,
MA, USA, #12-8898-82), APC anti-mouse Perforin antibody (BioLegend, San Diego,
CA, USA, #154304), and PE anti-mouse CD25 Antibody (BioLegend, San Diego, CA,
USA, #101904). Flow cytometric two-parameter dot plots and quadrant statistics
were generated using FACScan and CellQuest software (BD Biosciences, Mountain
view, CA, USA). For intracellular staining, cells were fixed and permeabilized
using Fix and Perm reagents (BD Biosciences, San Jose, CA) and incubated with
specific antibodies (God et al., 2015 ;
God et al., 2014 (link)). Briefly,
5×105 cells per group were re-suspended in 0.5 mL of
Fixation/Permeabilization Buffer and incubated at 2-8° C for 30 minutes.
After washing, the cell pellet was re-suspended in Permeabilization/Wash Buffer
and stained with CD4-PerCP, CD8-FITC, APC-perforin and PE-granzyme B. Cells were
then analyzed on FACScan using CellQuest software as described above.