Anti cd8 cy5 clone 53 6

Lymphocytes from thymus, lymph nodes, or spleen were stained in 100 μl of FACS buffer (PBS containing 5% FCS and 0.02% of NaN₂). The antibodies used were anti-CD3 PE (clone 145-2C11, eBioscience), anti-CD4 FITC (clone GK 1.5, eBioscience), anti-CD8 Cy5 (clone 53-6.7, eBioscience), anti-CD25 FITC (clone PC61, BioLegend), and anti-CD69 PE (clone H1.2F3, eBioscience). Cells were labeled with the appropriate concentration of conjugated antibodies for 1 h at 4°C as previously described (49 (link)). After labeling, cells were washed and analyzed. Cells were first gated for singlets (FSC-H vs. FSC-A) and lymphocytes (SSC-A vs. FSC-A). The lymphocyte gate was further analyzed for their expression of CD3, CD4, or CD8. In all the experiments, stained cells were acquired with FACScalibur flow cytometer and analyzed using FlowJo™ software (Tree Star, Inc., Oregon Corporation).

Lymphocytes collected from lymphatic organs (e.g. thymus, spleen) were stained in 100μl of FACS buffer (PBS containing 5% FCS and 0.02% of NaN₂). The antibodies used were anti-CD3 PE (clone 145-2C11, eBioscience), anti-CD4 FITC (clone GK 1.5, eBioscience), anti-CD8 Cy5 (clone 53-6.7, eBioscience), anti-CD25 FITC (clone PC61, BioLegend), anti-CD69 PE (clone H1.2F3, eBioscience). Cells were labeled with the appropriate concentration of conjugated antibodies for 1 h at 4 °C as previously described29 (link). After labeling, cells were washed and analyzed. In all experiments stained cells were acquired with FACScalibur flow cytometer and analyzed using FlowJo^TM software (Tree Star, Inc., Oregon Corporation).