7 aminoactinomycin d 7 aad and annexin 5

Approximately 1 × 10⁶ SPCs were washed in PBS and blocked for 10 min on ice with 1 μg mouse BD Fc block CD16/CD32 (BD Biosciences) in 100 µL FACS-buffer, PBS containing 0.1 % bovine serum albumin (BSA; Carl Roth), and then incubated with PE-conjugated anti-mouse-CD4 mAb (clone RM4–5, rat IgG2a, κ) as well as FITC-conjugated anti-mouse CD25 mAb (clone 3C7, rat IgG2b, κ) or their corresponding isotype controls (BD Biosciences) for 15 min at 4 °C in the dark. For further intracellular detection of the transcription factor Foxp3, cells were fixed, permeabilized and stained with rat-anti-FoxP3 antibody (clone FJK-16 s; eBioscience). Cell viability was determined with 7-aminoactinomycin D (7-AAD) and Annexin V (BD Biosciences). Cells were examined using a FACSCalibur™ flow cytometer (BD Biosciences) and data were analyzed with CellQuest™ Pro software (BD Biosciences).

J558 and BCL1 strains were purchased from ATCC, and M12.4.1 cells (31 (link)) were provided by Dr. Mark Boothby (Vanderbilt University, Nashville, TN, USA). J558 cells were cultured with DMEM (Welgene, Daegu, South Korea) supplemented with 10% horse serum (Welgene) and penicillin/streptomycin (Welgene). One million cells were transfected with 5 μg DNA by electroporation using Neon transfection system (Thermo Fisher Scientific) or NEPA21 Super Electroporator (Nepa Gene). Transfection efficiency was about 10–20%. Sixteen hours after transfection, the cells were harvested and subjected to further experiments. Cell viability was measured by the trypan blue exclusion method. To detect cell death, cells were stained with 7-aminoactinomycin D (7-AAD) and Annexin V (BD Biosciences) and assayed by flow cytometry.