Apc cy7 conjugated anti mouse cd4 antibodies

To examine the effects of the immunosuppressant treatments on immune cells, splenocytes were collected from mice and subsequently incubated with the appropriately diluted antibodies for 40 min at 4℃. Activated CD4⁺ effector T cells (Teffs) were stained with APC-Cy7-conjugated anti-mouse CD4 and FITC-conjugated anti-mouse CD44 antibodies, whereas CD4⁺ regulatory T cells (Tregs) were fixed/permeabilized after staining with CD4 antibody for intracellular PerCP-Cy5.5-conjugated anti-mouse forkhead box P3 (FOXP3) staining. Flow cytometry was performed using a fluorescence-activated cell sorting (FACS) Verse I or FACS Verse II flow cytometer (BD Biosciences, San Jose, CA, USA). Data were analyzed using FlowJo software, v10.0.7 (Tree Star, Inc., San Carlos, CA, USA). All experimental groups were compared to the negative control group. FITC-conjugated anti-mouse CD44 and PerCP-Cy5.5-conjugated anti-mouse FOXP3 antibodies and the Fixation/Permeabilization kit were purchased from eBioscience (San Diego, CA, USA). APC-Cy7-conjugated anti-mouse CD4 antibodies were purchased from BioLegend (San Diego, CA, USA).

For this study, 8- to 9-week-old male BALB/c (H-2^d) mice and C57BL/6 (H-2^b) mice were purchased from Orient Bio Inc. (Seongnam, Korea) and maintained according to the ethical guidelines of our institution.
The PE-Cy7-conjugated anti-mouse CD8, PerCP-Cy5.5-conjugated anti-mouse CD11c, PE-conjugated anti-mouse CD85k, FITC-conjugated anti-mouse CD44, PerCP-Cy5.5-conjugated anti-mouse FOXP3 antibodies, and the Fixation/Permeabilization kit were purchased from eBioscience (San Diego, CA, USA). APC-Cy7-conjugated anti-mouse CD4 antibodies were purchased from Biolegend (San Diego, CA, USA). TM, DX, and red blood cell lysis buffer, and Histopaque 1.083 were purchased from Sigma-Aldrich (St Louis, MO, USA). Mouse IL-6 enzyme-linked immunosorbent assay (ELISA) kit was purchased from BD Bioscience (San Jose, CA, USA).