Dilutions
Flow Cytometry: 1-4 µg/ml, Recommended protocol: 1) Perform staining of cell surface markers (CD25, CD4 etc.) for 20 min. at room temperature in the dark; 100 µl of peripheral blood. 2) Add 3 ml of PBS with 1% BSA, centrifugate at 300g and discard the supernatant. Further steps perform on ice and with ice-cold reagents. 3) Resuspend the cells in 5 ml of cold fixation solution (Miltenyi Biotec) and incubate for 30 min. on ice. 4) Centrifugate for 5 min. at 1000 g, 4°C, and discard the supernatant. 5) Resuspend the cells in 5 ml of ice-cold PBS with 1% BSA. 6) Centrifugate for 5 min. at 1000 g, 4°C, and discard the supernatant. 7) Resuspend the cells in 5 ml of ice-cold permeabilization solution (Miltenyi Biotec) and incubate 5 min. 8) Centrifugate for 5 min. at 1000 g, 4°C, and discard the supernatant. 9) Resuspend the cells in µl of ice-cold permeabilization solution and add 20 µl of FcR blocking solution and incubate for 5 min. (4°C, in the dark). 10) Perform Intracellular staining of Helios for 30 min. (4°C, in the dark) with 10 µl of anti-Helios PE antibody (22F6) in 100 µl. 11) Add 2 ml of ice-cold permeabilization solution and incubate for 5 min. at 4°C in the dark. 12) Centrifugate for 5 min. at 1000 g, 4°C, and discard the supernatant. 13) Resuspend the cells in 3 ml of ice-cold PBS with 1% BSA. 14) Centrifugate for 5 min. at 1000 g, 4°C, and discard the supernatant. 15) Resuspend the cells in 150 µl of ice-cold PBS with 1% BSA and measure on a Flow Cytometry device with appropriate setting as soon as possible. Keep cold until measuring.
