Trustain fcxtm anti mouse cd16 32

The collected leukocytes from peripheral blood, mesenteric LN, lung, and bone marrow were incubated with Fc-blocking TruStain FcX^TM (anti-mouse CD16/32, BioLegend, San Diego, CA, USA) antibody, followed by Zombie aqua (live/dead indicator, BioLegend, San Diego, CA, USA). The cells were stained with fluorescence-labeled antibodies. The following antibodies were purchased from BioLegend (San Diego, CA, USA): BV650-CD45.2 (104), Alexa Flour 700-CD3 (500A2), Pacific Blue-CD4 (RM4-5), BV570-CD8 (53-6.7), BV711-CD19 (6D5), and FITC-Gr-1 (RB6-8C5). All samples were acquired on a Cytek Aurora flow cytometry (Cytek Biosciences, Fremont, CA, USA), and the data were analyzed with FlowJo software (BD Bioscience, Franklin Lakes, NJ, USA). The calculation of the absolute number of leukocytes was carried out using CountBright^TM Absolute Counting Beads (Invitrogen, Waltham, USA) and according to the recommended manufacturer manual, with modifications. To calculate the absolute count, the following equation was applied: $$Absolute count \left(cells\right)=\frac{\left(Number of cell events\times assigned beads count of the lot\right)}{Number of bead events}\times \frac{1000 \mu \mathrm{L}}{The vol of 2\times {10}^6 cells}$$