Transcription factor phospho buffer set

At the National Institutes of Health, PBMCs were thawed in XVIVO media (Lonza) with 10% human AB serum (Sigma-Aldrich), pelleted, washed with XVIVO, and resuspended in XVIVO media with 1% human AB serum at the concentration of 10⁶ cells/ml. Then the cells were stimulated with 1,000 U IL-2 (Peprotech) for 10 min at 37°C, fixed with BD Fix/Lyse buffer (BD Biosciences) for 10 min at 37°C, and then washed with cold PBS with 0.2% BSA. Next, the fixed cells were permeabilized with −20°C methanol for 20 min on ice, washed five times with cold PBS with 0.2% BSA, and then stained with surface and intracellular flow cytometry antibodies for 30 min at 4°C. The fixed, permeabilized, and stained cells were washed with PBS and resuspended in PBS with 0.2% BSA for flow cytometry analysis. In Newcastle, thawed PMBCs were rested for 4 h in serum-free RPMI media. After the addition of surface markers and a fixable viability dye, 2 × 10⁵ cells were stimulated for 10 min at 37°C with 100 ng/ml of IL-2, IL-7, or IL-15, or left unstimulated. The Transcription Factor Phospho Buffer set (BD Biosciences) was used to fix and permeabilize cells according to the manufacturer’s instructions. Cells were stained with the remaining surface as well as intracellular markers for 45 min at 4°C before cells were washed in TFP Perm/Wash buffer and finally resuspended in FACS buffer for acquisition.

For immune cell profiling using CyTOF, blood was collected in BD Vacutainer ACD tubes from five patients undergoing splenectomy. Prior to storage at −80°C, RBCs were lysed from whole blood using the BD Biosciences Lyse/Fix kit according to the manufacturer’s protocol. To decrease intrasample variability and allow analysis of cryosensitive granulocytic cells, all blood samples were prepared and cryopreserved immediately after collection. This precluded the use of a Live/Dead discriminator dye. Cells were thawed and stained individually with DNA-intercalating barcodes using the Fluidigm Barcoding Kit for simultaneous sample processing, as previously described (22 (link)). Cells were mixed and stained together for 30 min at room temperature with metal-conjugated cell surface Abs. The cells were then permeabilized using the BD Biosciences Transcription Factor Phospho Buffer Set according to the manufacturer’s protocol. Following permeabilization, the cells were stained with the intracellular Abs, followed by DNA intercalator to identify cellular events.

CD19 CAR T cells produced from peripheral blood mononuclear cells (PBMCs) of an HD and untransduced PBMCs from three different HDs were plated at 1×10⁶ cells per well in a 96-well plate. The plate was spun at 500× g for 5 minutes, and cells were incubated in flow cytometry blocking buffer (1× PBS containing 10% human serum and 10% mouse serum) for 10 minutes at room temperature. Cells were washed with flow cytometry wash buffer (1× PBS containing 2% FBS) and incubated with the following antibodies for 1 hour at 4°C: CD66 (B1.1/CD66), CD3 (UCHT1), CD4 (SK3), CD8 (SK1), and CD25 (2A3) from BD Biosciences, and LAG-3 (11C3C65), PD-1 (EH122H7), and TIM-3 (F382E2) from Biolegend (San Diego, CA, USA). After washing, cells were fixed and permeabilized with Transcription Factor Phospho Buffer Set (BD Biosciences) according to the manufacturer’s instructions. After washing, cells were then stained intracellularly with the following antibodies for 1 hour at 4°C: CTLA-4 (I4D3) from BD Biosciences, FOXP3 (150D) and Tbet (4B10) from Biolegend, and EOMES (WD1928) from Thermo Fisher Scientific. Samples were analyzed by flow cytometry on a BD LSRFortessa X-20 instrument with a minimum number of 50,000 cells per sample analyzed and FlowJo Software (FlowJo LLC).