Bv605 anti mouse cd4

For flow cytometry analysis, single-cell suspensions were stained with the following antibodies: BV605 anti-mouse CD4 and Alexa Fluor 700 CD8 (Biolegend, San Diego, CA, USA). For intracellular staining, cells were stimulated for 4 h at room temperature with PMA (50 ng/mL), ionomycin (1 µg/mL), and Brefeldin A. Following fixation and permeabilization, cells were stained with Alexa Fluor 647 anti-human IFN-γ and PE anti-human TNF-α (Biolegend, San Diego, CA, USA). Cells were acquired with a NovoCyte Quanteon (Agilent Technologies, Santa Clara, CA, USA), and data analysis was performed with FlowJo software (Version 10.8, FlowJo, Ashland, OR, USA).

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).