Fitc anti mouse cd45 antibody

Ba/F3 P210 BCR-ABL cells (5 × 10⁶ cells) were administered via intravenous (i.v.) injections into the lateral tail veins of immunosuppressed mice (n = 12), on days 11, 14, and 17 (Fig. 1), thereafter called leukemia mice (n = 12). To determine the presence of Ba/F3 BCR-ABL P210 cells in mice peripheral blood, we used the presence of both CD45 (a leukocyte marker, most expressed in lymphocytes) and F4/80 surface (https://cell.brc.riken.jp/en/rcb/baf3; Last update: 2022.07.25), and ABL proteins according to supplier’s recommendation. Briefly, total blood was collected, and 30 μl of the sample was incubated with rat anti-mouse FITC-CD45 (FITC anti-mouse CD45 Antibody, Cat# 103108, Biolegend), rat anti-mouse PE-F4/80 (PE anti-mouse F4/80 Antibody, Cat# 123110, BioLegend), and anti-ABL (c-Abl Antibody; Cat# MA5-14398; Invitrogen) antibodies for 15 min. Then, samples were incubated in red blood cell lysis solution for 30 min at 37 °C. Finally, the sample was centrifuged at 2000 rpm for 10 min. and incubated with DyLight 488 donkey anti-mouse antibody (1:500) (to identify ABL primary antibody), rinsed and resuspended for analysis on a BD LSR Fortessa II flow cytometer (BD Biosciences). Fifty thousand events were acquired, and the acquisition analysis was performed using FlowJo 7.6.2 Data Analysis Software.

After the isolation of stromal vascular fraction of PVAT, cells were suspended in PBS containing 5% FBS. Zombie NIR Fixable Viability Kit (BioLegend) was used to label dead cells according to the manufacturer’s instructions. Nonspecific binding of the antibodies to Fc receptors was blocked by using Mouse Fc receptor-blocking agent (BioLegend). Cells were incubated at room temperature 30 mins with FITC anti-mouse CD45 Antibody (BioLegend, #103107, 1:50), PE/Cyanine7 anti-mouse F4/80 antibody (BioLegend, #123113, 1:50), PE anti-mouse CD11b antibody (BioLegend, #101207, 1:50), APC anti-mouse CD206 antibody (BioLegend, #141707, 1:50)‚ and Brilliant Violet 421 anti-mouse CD11c antibody (BioLegend, #117329, 1:50), then centrifuged and fixed with PBS containing 2% PFA. Cells were analyzed using a FACSMelody cell sorter (BD Biosciences). For the analysis of flow cytometry data, CD45⁺, CD11b⁺, and F4/80⁺ cells were defined as macrophages, which could be further differentiated into classically (M1; defined as CD11c⁺, CD206⁻) and alternatively activated (M2; defined as CD11c⁻, CD206⁺) macrophages^{77 (link)}. Data analyses were performed using FlowJo software (Tree Star).