FoxP3 Antibody, anti-mouse, PE, REAfinity™

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Isolation of splenocytes was done by mashing the spleen through a 70-μm cell strainer. The cells were washed with phosphate-buffered saline and after spinning at 300g for 6 minutes, the cell pellet was resuspended in 1 mL lysis buffer for 3 minutes. Afterward, the cells for cytokine staining were washed with RPMI-1640 +10% fetal calf serum; 1 × 106 cells/well were plated in a 24-well plate and activated with stimulation cocktail for 4 hours.	After incubation cells were washed with fluorescence-activated cell sorting buffer and stained with surface antibodies

Upstream tips
Isolation of splenocytes was done by mashing the spleen through a 70-μm cell strainer. The cells were washed with phosphate-buffered saline and after spinning at 300g for 6 minutes, the cell pellet was resuspended in 1 mL lysis buffer for 3 minutes. Afterward, the cells for cytokine staining were washed with RPMI-1640 +10% fetal calf serum; 1 × 106 cells/well were plated in a 24-well plate and activated with stimulation cocktail for 4 hours.
Protocol tips
After incubation cells were washed with fluorescence-activated cell sorting buffer and stained with surface antibodies

Publication protocol

"Isolation of splenocytes was done by mashing the spleen through a 70-μm cell strainer. The cells were washed with phosphate-buffered saline and after spinning at 300g for 6 minutes, the cell pellet was resuspended in 1 mL lysis buffer for 3 minutes. Afterward, the cells for cytokine staining were washed with RPMI-1640 +10% fetal calf serum; 1 × 106 cells/well were plated in a 24-well plate and activated with stimulation cocktail for 4 hours. After incubation cells were washed with fluorescence-activated cell sorting buffer and stained with surface antibodies CD4, CD3, and CD19. The cells were then fixed and permeabilized. Next anti-cytokine antibodies were added (IFN-gamma, IL10, IL17, and IL4) and incubated at room temperature for 20 minutes.
Staining for activation marker was done 1 × 106 cells per tube. For blocking of nonspecific Fc-mediated interactions splenocytes were pre-incubated with 1 μg of anti-mouse CD16/CD32 for 5 minutes. Thereafter, the antibody cocktail (CD4, CD3, CD11b, Ly6G, CD69, CD25 and CD62L) was added and incubated at 4°C for an additional 20 minutes and then analyzed by flow cytometry.
For intracellular staining of nucleus transcription factors GATA-3, FoxP3, and Tbet 1 × 106 splenocytes were not activated and directly treated with anti-mouse CD16/CD32 and then stained for the surface marker CD4, CD69, and CD25. After fixation and permeabilization cells were stained for the transcription factors (FixPerm True-Nuclear Transcription Factor Buffer Set, 424401; BioLegend)."

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