Mouse anti cat cd8 alpha beta rpe

Cells were washed with complete RPMI medium and diluted to a concentration of 1 × 10⁶ cells/100 μL. Cells in the 2 aliquots (100 μL/aliquot) were pelleted by centrifugation at 1800g for 8 minutes and incubated with 100 μL of PBS solution and optimal concentrations of fluorescein isothiocyanate‐conjugated CD4⁺ (Mouse anti‐cat CD4:FITC, Bio‐Rad, Hercules, California) and phycoerythrin‐conjugated CD8⁺ (Mouse anti‐cat CD8 alpha/beta:RPE, Bio‐Rad) for 15 minutes at 4°C in the dark. Concentration of the antibodies was 1 μg of IgG/10 μL of PBS solution. Cells were washed twice with 200 μL of flow cytometry buffer (97% PBS solution and 3% heat‐inactivated fetal bovine serum) followed by centrifugation at 1800g for 4 minutes. Cells were resuspended in flow cytometry buffer, and lymphocytes were gated for characteristic forward‐ and side‐scatter profiles. We collected 25 000 total events per sample for flow analysis. The percentage of cells stained with antibody against CD4⁺ and CD8⁺ was determined as 2‐color flow cytometry profiles (BD FACSCalibur, BD Bioscience, San Jose, California). Percentages of stained cells were calculated by the use of flow cytometer software (FCS Express 4, BD Bioscience).

Each whole blood sample (3 mL) was immediately mixed with EDTA to prevent blood clotting. After this, 50 µL of the blood mixture was transferred to a 5 mL round-bottomed polystyrene tube (Falcon^®, Corning, NY, USA) and mixed with monoclonal antibodies against feline CD4⁺ (10 µL; mouse anti-cat CD4⁺: FITC, Bio Rad Laboratories, Singapore) and CD8⁺ (10 µL; mouse anti-cat CD8+ alpha/beta: RPE, Bio Rad Laboratories, Singapore). After vortexing, the mixture was incubated in a dark room for 15 min. Then, 1000 µL of 1% lysis buffer (BD FACS™ Lysing Solution, San Jose, CA, USA) was added to lyse the red blood cells. The mixture was centrifuged at 4 °C and 1500 rpm for 5 min. The pellet was washed with 3000 µL of phosphate-buffered saline pH 7.4 (Sigma-Aldrich, Saint Louis, MO, USA) and centrifuged at 4 °C and 1500 rpm for 5 min. The cell pellet was reconstituted and fixed with 200 µL of 1% paraformaldehyde (Sigma-Aldrich) and stored in a dark room until analysis using the flow cytometer (BD FACSCalibur™, San Jose, CA, USA). The analysis of the lymphocyte subset (CD4⁺ and CD8⁺) used the flow cytometer at the King Chulalongkorn Memorial Hospital, Bangkok, Thailand. The ratio was considered to be decreased when it was <1.