Anti mouse cd45 pacific blue

To isolate adult endothelial cells from mouse kidney, liver, heart, and lung, mice were injected intravitally with 25 μg of anti-VE-cadherin-AF647 antibody (clone BV13, Biolegend) retro-orbitally in 6–8-week-old male C57BL/6J mice under anesthesia 10 min before they were killed and the organs harvested. For cell sorting, organs were minced and incubated with Collagenase A (25 mg ml⁻¹) and Dispase II (25 mg ml⁻¹) at 37 °C for 20–30 min to create a single-cell suspension. Cells were filtered through a 40-μm filter immediately before counter staining. The single-cell suspension was first blocked with an Fc-quenching antibody before antibody staining with anti-mouse CD31-Alexa Fluor® 488 (102414, Biolegend), anti-mouse CD45-Pacific Blue™ (103126, Biolegend), and anti-mouse Podoplanin-PE/Cy7(127412, Biolegend). Embryonic tissues were dissected and processed through the same antibodies. Following staining, cells were processed for FACs sorting.

Lung leukocytes (1 × 10⁶) were stimulated with CD3/CD28 anti-mouse antibodies (2.5 μg/mL ea.) in the presence of 1X brefeldin A (Biolegend) for 8–12 h prior to surface staining with anti-mouse CD45 Pacific blue (clone; S18009F, Biolegend, San Diego, CA, USA), anti-mouse CD3 APC (clone; 145-2C11, Tonbo Biosciences, San Diego, CA, USA), anti-mouse CD8 FITC (clone; 53–6.7, Biolegend, San Diego, CA, USA), anti-mouse CD4 BV605 (clone; GK 1.5, Biolegend, San Diego, CA, USA), anti-mouse CD69 PE/Cy7 (clone; H1.2F3, Invitrogen, CA), and anti-mouse CD103 BV711 (clone; BV711, Biolegend, San Diego, CA, USA). After two washes, cells were fixed in 2% paraformaldehyde and treated with permeabilization buffer (0.5% tween 20 in FACS buffer). Cells were stained with anti-mouse GzmB PE (clone QA18A28; Biolegend, San Diego, CA, USA) and anti-mouse IFN-γ APC-Cy7 (clone; B-XMG1.2, Biolegend, San Diego, CA, USA).

Spleen cells were prepared as described previously [18 (link)]. Briefly, the spleen was mechanically dissociated and passed through a 70um filter. The final 4 ml suspension was layered onto 2 ml Ficoll-Paque (GE,17–1440-02) and the cells at the interface were collected after certification (500 g,20 min,4 °C). Single cell samples were incubated with antibodies to surface antigens for 30 minutes on ice at 4 °C in the dark. Fluorochrome compensation was performed with single-stained UltraComp eBeads. Flow cytometery was performed on the BD LSRFortessa flow cytometer (BD biosciences). Data analysis were performed using Flowjo software.
The antibodies used for profiling splenic T cells included anti-mouse CD45-Pacific Blue (1:200; BioLegend, 103,126); anti-mouse CD3e-FITC (1:200; BD Pharmingen, 553,062); anti-mouse CD4-APC-Cy7 (1:200; BD Pharmingen, 552,051); anti-mouse CD8-Percp (1:200; BioLegend, 100,732); anti-mouse CD44-V500 (1:200; BD Pharmingen, 560,781); anti-mouse CD62L-BUV395 (1:200; BD Pharmingen, 740,218); anti-mouse CCR2-PE (150609).