Pacific blue anti mouse gr 1

Cells were pelleted and washed in phosphate-buffered saline (PBS) supplemented with 2% FBS (2% FBS/PBS). The cell suspension was first blocked with TruStain fcX (anti-mouse CD16/32) antibody (BioLegend) for 5 min, and then stained with the following antibodies (Abs) for 15 min at 4°C: APC anti-mouse CD11b, Pacific Blue anti-mouse Gr-1, PE anti-mouse F4/80, FITC anti-mouse CD11c, APC-Cy7 anti-mouse Ly-6C, FITC anti-mouse Ly-6G, Pacific Blue anti-mouse CD4, and FITC anti-mouse CD8α (BioLegend). Next, the cells were washed and resuspended in 2% FBS/PBS. Shortly before performing measurements, a 7-amino actinomycin D viability staining solution (BioLegend) was added to each sample to stain dead cells. Flow cytometry analysis was performed on a BD FACSCanto II flow cytometer (BD Biosciences), and results were analyzed using the FlowJo software (version10.7.0, BD Biosciences).

Cells were pelleted, washed with 2% FBS/HBSS, blocked with TruStain fcX (anti-mouse CD16/32) antibodies (BioLegend, CA, USA) for 5 min, and then stained with the following antibodies for 15 min at 4°C: APC anti-mouse CD11b, Pacific Blue anti-mouse Gr-1, APC-Cy7 anti-mouse Ly-6C, FITC anti-mouse Ly-6G, APC anti-mouse CD3ε, Pacific Blue anti-mouse CD4, PE anti-mouse NK1.1, and FITC anti-mouse CD8α (BioLegend). The cells were then washed and resuspended in 2% FBS/HBSS. Shortly before performing measurements, a 7-amino actinomycin D viability staining solution (BioLegend) was added to each sample to stain dead cells. Flow cytometry analysis was performed using a BD FACSCanto II flow cytometer (BD Biosciences, NJ, USA). Data were analyzed using the FlowJo software (version 10.7.0, BD Biosciences). The gating strategy used for flow cytometry analysis was as follows: monocytes (7AAD⁻CD45⁺CD11b⁺Ly-6G⁻Ly-6C^hi), neutrophils (7AAD⁻CD45⁺CD11b⁺Ly-6G⁺Ly-6C^int), CD4⁺ T cells (7AAD⁻CD45⁺CD3ε⁺CD4⁺NK1.1⁻), CD8⁺ T cells (7AAD⁻CD45⁺CD3ε⁺CD8α⁺NK1.1⁻), and NK cells (7AAD⁻CD45⁺CD3ε⁻NK1.1⁺) (Figure S1A).