Staining of Nuclear Targets by Flow Cytometry

1、Add an appropriate amount of antibody for surface staining to 100 μl anticoagulant, according to the experimental protocol and product instructions. Shake well and incubate at room temperature avoiding light for an appropriate time.
2、Add 2 ml 1× FCM Lysing Solution (Cat No. LYS01) working solution to each tube, vortex and shake well. Incubate at room temperature avoiding light for 15 minutes. Centrifuge at 300-400 × g for 5 minutes at room temperature and remove the supernatant.
3、Add 2 ml 1× Flow Cytometry Staining Buffer (Cat No. S1001) to each tube, vortex and shake well. Centrifuge at 300-400 × g for 5 minutes at room temperature and remove the supernatant.
4、Prepare the Fixation/Permeabilization Concentrate (4×) (Cat No. ICC01) and Fixation/Permeabilization Diluent (Cat No. ICD01) into a 1× working solution in a 1:3 ratio. Stain in flow cytometry tubes, requiring 1 ml per sample. Do not exceed 1 day of storage. Dilute the Permeabilization Buffer (10×) (Cat No. ICB01) with distilled water to make a 1× working solution. Stain in flow cytometry tubes, requiring 8.5 ml per sample. The remaining solution can be stored for up to 1 week at 2-8℃.
5、Add 1 ml Fixation/Permeabilization working solution to each tube, vortex and shake well. Incubate at room temperature avoiding light for 30-60 minutes.
6、No washing is required. Add 2 ml 1× Permeabilization Buffer to each tube. Centrifuge at 300-400 × g for 5 minutes at room temperature and remove the supernatant.
7、(Optional) Repeat step 6.
8、Resuspend the pellet in 100 μl 1× Permeabilization Buffer. Usually, the residual liquid after washing is sufficient.
9、(Optional) Add 2 μl normal rat serum (Cat No. NRS500) to the cell suspension for blocking. Incubate at room temperature avoiding light for 15 minutes.
10、No washing is required. Add an appropriate amount of FoxP3 antibody or other suitable antibodies and the same amount of isotype control according to the instructions. Shake well and incubate at room temperature avoiding light for at least 30 minutes.
11、Add 2 ml 1× Permeabilization Buffer to each tube. Centrifuge at 300-400 × g for 5 minutes at room temperature and remove the supernatant.
12、(Optional) Repeat step 11.
13、Resuspend in 500 μl 1× Flow Cytometry Staining Buffer (Cat No. S1001) and detect.