CD25 Monoclonal Antibody (PC61.5), APC, eBioscience™

Protocol tips

Upstream tips	Protocol tips	Downstream tips
For Treg cell staining, we used anticoagulant in whole blood for 200 μL per tube, and after lysis by Red Blood Cell Lysis Buffer (Beijing Solarbio Science and Technology Co., Beijing, China), cells were resuspended in PBS.	mixing and incubation at room temperature in the dark for 30 min. After washing 2 times, cells were fixed and permeabilized, the we added 10 μL Foxp3-PE (PE anti-mouse Foxp3, eBioscience, USA), followed by incubation for 30 min at room temperature.

Upstream tips
For Treg cell staining, we used anticoagulant in whole blood for 200 μL per tube, and after lysis by Red Blood Cell Lysis Buffer (Beijing Solarbio Science and Technology Co., Beijing, China), cells were resuspended in PBS.
Protocol tips
mixing and incubation at room temperature in the dark for 30 min. After washing 2 times, cells were fixed and permeabilized, the we added 10 μL Foxp3-PE (PE anti-mouse Foxp3, eBioscience, USA), followed by incubation for 30 min at room temperature.

Publication protocol

For Treg cell staining, we used anticoagulant in whole blood for 200 μL per tube, and after lysis by Red Blood Cell Lysis Buffer (Beijing Solarbio Science and Technology Co., Beijing, China), cells were resuspended in PBS. Then, we added 10 μL each CD4-FITC (FITC anti-mouse CD4, eBioscience, USA) and CD25-APC (APC anti-mouseCD25, eBioscience), followed by mixing and incubation at room temperature in the dark for 30 min. After washing 2 times, cells were fixed and permeabilized, the we added 10 μL Foxp3-PE (PE anti-mouse Foxp3, eBioscience, USA), followed by incubation for 30 min at room temperature.

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