Alexa fluor 700 rat anti mouse cd8a

For flow cytometry analysis, whole blood samples were stained following the almost identical protocol already described elsewhere^{72 (link)} by using the following fluorochrome-labeled anti-mouse mAbs: FITC Rat Anti-Mouse CD45; APC Rat Anti-Mouse Ly-6G; PE-CF594 Rat Anti-Mouse Ly-6G and Ly-6C; PE-Cy7 Rat Anti-Mouse CD4; Alexa Fluor 700 Rat Anti-Mouse CD8a; APC-Cy7 Rat Anti-Mouse CD45R; PE Hamster Anti-Mouse CD3e and PerCP-Cy5.5 Rat Anti-Mouse CD335 (BD Biosciences, San Diego, USA). In brief, protected from light, blood was incubated for 15 min at room temperature. The stained samples were then treated with BD FACS lysing solution (BD Biosciences) following manufacturer’s instructions. Thereafter, white blood cells were washed twice with washing buffer (PBS, 1% BSA and 0.1% sodium azide), resuspended in measuring buffer (PBS, 0.1% BSA and 0.1% sodium azide) and measured by flow cytometry using LSR Fortessa (BD Biosciences, San Diego, USA). Plots were compensated and analyzed by FlowLogic Software 7.2.1 (Inivai Technologie, Mentone Victoria, Australia). Gating strategy is shown in Supplemental Fig. 1.

Lymphocytes were suspended in 200 μL of FACS buffer (PBS with 5% FBS) in FACS tubes. To each tube, a mixture of monoclonal antibodies, including APC Cy7 rat anti-mouse CD4 (552051; BD Biosciences, San Diego, CA, USA), FITC rat anti-mouse CD25 (553071; BD Biosciences, San Diego, CA, USA), PE rat anti-mouse CD3 (555275; BD Biosciences, San Diego, CA, USA), and Alexa Fluor 700 rat anti-mouse CD8a (557959; BD Biosciences, San Diego, CA, USA), was added, and the cells were incubated at 4 °C for 15 min. Then, the cells were washed with FACS buffer and fixed with 2% paraformaldehyde. For intracellular staining, after washing, cells were permeabilized with ice-cold methanol at room temperature for 20 min. After washing, the cells were incubated at 4 °C for 20 min with Alexa Fluor 647 rat anti-mouse FoxP3 (560401; BD Biosciences, San Diego, CA, USA). After washing, the stained cells were analyzed on BD LSR Fortessa Cell Analyzer equipped with DIVA software. Cells were gated as follows: The lymphocyte population was gated on the FSC/SSC channel and was further gated for the CD3⁺ population, which was then further gated into CD4 and CD8 populations. From the CD3⁺CD4⁺ cells, the CD25⁺ population was taken as a parent for FoxP3 assignment. Each sample was prepared in triplicate, and 50,000 events were collected.