Fitc rat anti mouse cd8a

Immune cells from animal tissue or blood were separated by using HISTOPAQUE^®‐1077(10771, Sigma), and then the dissociated lymphocytes were resuspended in PBS and stained for surface markers for 20 minutes with antibodies: PerCP‐Cy^TM 5.5 rat anti‐mouse CD45 (550994, BD Biosciences, 1:200), FITC rat anti‐mouse CD8a (553030, BD Biosciences, 1:500), APC rat anti‐mouse CD11b (553312, BD Biosciences, 1:200), PE rat anti‐mouse F4/80 (565410, BD Biosciences, 1:200), APC anti‐mouse CD206 (MMR) (141707, BD Biosciences, 1:200), APC mouse anti‐human HLA‐ABC (555555, BD Biosciences, 1:200), APC mouse anti‐human HLA‐DR (560896, BD Biosciences, 1:125), and BV421 rat anti‐mouse CD119 (740032, BD Biosciences, 1:200). Flow cytometry data were obtained on an LSRFortessa, and data analysis was achieved through FlowJo software (Tree Star).

Flow cytometry was performed to detect the proportion of CD4⁺ or CD8⁺ lymphocytes in the spleen. Following sacrifice, the spleens were removed under sterile conditions, weighed, and processed into single cell suspensions, which then stained simultaneously with the following antibodies: PerCP-Cy^™5.5 Rat Anti-Mouse CD3 (BD Biosciences, 560527), BV421 Rat Anti-Mouse CD4 (BD Biosciences, 562891), and FITC Rat Anti-Mouse CD8a (BD Biosciences, 553030), protected from light for 30 min. Labeled cells were fixed with 1% PFA and analyzed with a LSRFortessa X-20 flow cytometer (BD Biosciences, MA, United States) on FACSDiva 8.0.1 software (BD Biosciences). For each sample, corresponding isotype control antibodies were used. CD3⁺ cells were gated as T lymphocytes, and then the CD4⁺ and CD8⁺ populations were analyzed.