Pe conjugated anti mouse cd8 antibody

The percentage of CD4+ and CD8+ T lymphocyte from the mouse spleen was analyzed by flow cytometry, as described in previous studies with minor modifications [26 (link),27 (link)]. Briefly, spleen lymphocytes were isolated as described above and fixed with 1% formaldehyde solution. Subsequently, the lymphocytes were treated with FITC-conjugated anti-mouse CD4 antibody, phycoerythrin (PE)-conjugated anti-mouse CD8 antibody, FITC mouse IgG2b κ isotype control antibody, or PE mouse IgG1 κ isotype control antibody (BioLegend, San Diego, CA, USA) at 4°C for approximately 2 h following the instructions of manufacturer. The labeled cells were washed with cold PBS solution three times and analyzed by a flow cytometer (BD CantoII, Franklin Lakes, NJ, USA). All treatments were performed in triplicate.

The grind-sieve method was used to isolate CD8⁺ T cells from the TDLNs (from the lateral fold to the thigh muscle tissue, close to the deep iliac circumflex branch, n = 3) of mice of different ages²⁶ (link). The TDLNs were dissected and placed in a centrifuge tube containing 1 mL PBS. The thymus tissue was disintegrated by repeated scraping over a metal grid, tissue debris was removed through a mesh cell screen (5 cm × 5 cm); the filtrate was mixed with an equal volume of red blood cell lysate and paced at 4 °C for 5 min. It was then centrifuged at 1000×g for 4 min to collect the cells, and subsequently, 200 μL of PBS containing 1 μL PE-conjugated anti-mouse CD8 antibody (Bio-Legend, B266455, San Diego, CA, USA) and 1 μL FITC-conjugated anti-mouse CD3 antibody (Bio-Legend, B274724, San Diego, CA, USA) were added and the sample incubated for 30 min. The total number of isolated cells in the TDLNs was counted using a hemocytometer, and the variation in CD8⁺ T cells in the isolated cell samples was determined by flow cytometry.
The grind-sieve method was used to isolate T lymphocytes from the thymus of 2-, 8-, and 16-month-old mice (n = 3). Using the above isolation method for T lymphocytes, CD3⁺ T cells in the thymus were labeled and measured.