Ic fixation kit

BD LSRII Fortessa flow cytometer was used to acquire cells. Cells were stained with the following monoclonal antibodies anti- H-2K^d (SF1–1.1.1), CD4 (RM4–5/GK1.5), CD8α (53–6.7), CD8β (H35–17.2), CD25 (PC61.5), Lag3 (C9B7W), PD-1 (J43), Tim3 (RMT3–23), Foxp3 (FJK-16s), CCR9 (CW-1.2), α4β7 (DATK-32), IFN-γ (XMG1.2), IL-17 (eBio17B7) and TNF-α (MP6-XT22). Antibodies were purchased either from Thermo Fischer Scientific (Waltham, MA) and Biolegend (San Diego, CA). Cells were stimulated with cell stimulation cocktail and protein transport inhibitor cocktail (Thermo Fischer Scientific, Waltham, MA) for 5 hours to measure intra-cellular cytokines. For intracellular staining, cells were fixed using the Foxp3/Transcription factor staining buffer set or IC fixation kit (Thermo Fischer Scientific, Waltham, MA). Active caspase-3 was measured by CaspGLOW™ Fluorescein Active Caspase-3 Staining Kit (Thermo Fischer Scientific; Waltham, MA). Cells were stained with Fixable Viability Dye ef780 (Thermo Fischer Scientific, Waltham, MA) to exclude dead cells for all experiments. Data were analyzed with FlowJo (Tree Star Inc., Ashland, OR).

Ten days after transduction, library-expressing OCI-AML2 cells were treated with DMSO or 200nM selinexor. After 48-hours, cells were pelleted, washed with 1X PBS and fixed/permeabilized using the Thermo IC Fixation Kit. Fixation, permeabilization and staining was performed according to manufacturer’s instruction with slight modification; fixed 35E6 cells/15mL tube (buffers scaled) at R.T. for 15 minutes , washed with 1X Permeabilization Buffer followed by wash with FACS buffer (1X PBS, 2% FBS, 0.1% Sodium Azide, 2mM EDTA). Cells were stained in 1X Permeabilzation Buffer with a 1:200 dilution of p-AKT T308 (244F9) (CST #4056) primary antibody (1mL of Permeabilzation Buffer/primary antibody solution per 35E6 cells) at R.T. for 2 hours with gentle rocking. Cells were again washed with 1X Permeabilization Buffer followed by FACS buffer and stained in 1X Permeabilzation Buffer with 1:200 dilution of Alexa Fluor 488 Conjugate (CST #4412) secondary antibody at R.T. for 1 hour with gentle rocking. Cells were washed with 1X Permeabilization Buffer followed by FACS buffer and resuspended at 25E6cells/mL in FACS buffer in preparation for FACS analysis. All centrifugations performed at 700g.