Fitc rat anti mouse cd25

Lymphocytes were labeled with APC Cy7 rat anti-mouse CD4 (Cat. No. 552051; BD Biosciences, San Diego, CA, USA), PE rat anti-mouse CD3 (555275; BD Biosciences, San Diego, CA, USA), and FITC rat anti-mouse CD25 (553071; BD Biosciences, San Diego, CA, USA) for 15 min at 4 °C. Then, the cells were washed with FACS buffer (PBS with 5% FBS) and fixed with 2% paraformaldehyde. Next, the lymphocytes were permeabilized with ice-cold methanol at room temperature (RT) for 20 min, washed, and labeled with Alexa Fluor rat anti-mouse Foxp3 (560401; BD Biosciences, San Diego, CA, USA). The cells were examined on a BD LSRFortessa cell analyzer (BD Biosciences, San Jose, CA, USA), and the obtained data were analyzed using BD FACSDIVA software version 7.0 (BD Bioscience, San Jose, CA, USA) [44 (link)].

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.