Bcl 2 pe

Fresh total cells from the respective tissues were directly stained with the following monoclonal antibodies (mAbs) at predetermined optimal dilutions for 20 min at 4°C: CD3-PE, CD8-Alexa700, CD4-HorizonV500, CD127-FITC, and CD25-PeCy7 (eBioscience). Intracellular detection of FOXP3 (FOXP3-E450, eBioscience), CTLA-4 (CTLA-4-APC, eBioscience), Ki-67 (Ki-67-FITC, BD Pharmingen), and Bcl-2 (Bcl-2-PE, BD Pharmingen) was performed on fixed and permeabilized cells using appropriate buffer (eBioscience) (incubation 30 min at 4°C). Cells were acquired on an LSR II flow cytometer (Becton Dickinson) and analyzed using FlowJo software (Tree Star, Inc.). Dead cells were excluded by forward/side scatter gating.

The expanded cells were subjected to phenotypic characterization using a panel of anti-bodies. For the detection of intracellular proteins the cells were permeabilized using a BD fixperm kit (BD Pharmingen, San Jose, CA, USA) and the staining was done according to the manufacturer’s instructions. Isotype matched antibodies were used as controls. The fluorescently labeled cells were acquired on FACS Canto II and Aria (BD, San Jose, CA, USA) and data were analyzed by FACS DIVA - version 5.0 The details of the antibodies used are as follows: annexin V FITC, CD34 APC/PE, CD38 FITC, Bcl-2 PE, Bax FITC, CD33 FITC/PE, CD19 APC CD3 FITC, CD61 APC, CD45 PE/PE-CY-7 murine CD45.1 Pacific blue (BD Pharmingen) and CD133 PE (Milteny Biotech, Colonge, Germany).

PBMC were isolated and cryopreserved following standard procedures [33 (link)], then shipped to the FHCRC laboratory in Seattle for later analysis. To evaluate T-cell activation, cryopreserved PBMC were thawed in R10 (RPMI 1640 supplemented with 2mML-glutamine, 25mM HEPES (Invitrogen) 10% FBS (Benchmark), 50μg/ml streptomycin, 50U/ml penicillin (Invitrogen), and 50U/ml benzonase (Novagen), and then washed and stained immediately. Sample viability was determined by LIVE/DEAD Aqua Cell Stain (Invitrogen). Surface staining used integrin β7 Phycoerythrin Cyanine (PECy) 5, Cluster of differentiation (CD) 49d (α4) Allophycocyanin (APC), C-C chemokine receptor type 5 (CCR5) PECy7, Cutaneous Leukocyte Antigen (CLA) biotin, CD14 V450 (all Becton Dickinson [BD] Biosciences), and CD103 (αE) Peridinin Chlorophyll Protein Cyanine (PerCPCy) 5.5 (Acris Antibodies). Intracellular staining used CD3 Ax700, Ki-67 Fluorescein Isothiocyanate FITC, B cell lymphoma 2 (Bcl-2) PE, Streptavidin APC-Cy7 (all BD), and CD4 Energy Coupling Dye ECD (Beckman Coulter). Flow cytometric analysis was performed using an LSR II flow cytometer (BD); post-acquisition analysis used FlowJo software (Tree Star, Inc). Personnel involved in thawing, staining and acquiring flow cytometric data were blinded from samples’ pre/post procedure status.

Patient-derived JMML cells or mechanically-dissociated mouse tissue cells were surface stained with monoclonal antibodies from Biolegend and Becton Dickinson: CD45-mouse Horizon Blue V605 (30-F11), CD45-human PE (HI30), CD11b AlexaFluor488 (M1/70), CD38 PerCP/Cy5.5 (HIT2), CD34 PE/Cy7 (581), CD33 APC (WM53), CD13 APC/Cy7 (WM15), CD14 Pacific Blue (M5E2), CD19 FITC (HIB19), CD66b PerCP/Cy5.5 (G10F5), CD3 PE/Cy7 (SK7), CD71 APC/Cy7 (CY1G4), CD235a BV421 (GA-R2). For intracellular staining, live cells were first stained for surface proteins and then fixed, permeabilized, and stained for intracellular proteins using the Foxp3 Transcription Factor Staining Buffer Set (Invitrogen) according to the manufacturer’s instructions. For intracellular staining, the following antibodies were used: BCL-2 PE (3F11 BD), MCL-1 PE (Y37 Abcam), BCL-XL FITC (H-5 Santa Cruz) and matching isotype controls. Normalized median fluorescence intensity (MFI) was calculated according to the following equation: normalized MFI = (MFI of protein of interest) − (MFI of each isotype). Flow cytometry was performed using a BD LSRFortessa; for analyses FlowJo software was used. The gating strategy is shown in Online Supplementary Fig. S1.

PBMCs, isolated as described above, were resuspended to 1×10⁶ cells/ml in PBS. The cells were surface-stained with CD3-BV786, CD38-BUV737, HLA-DR-PE, CCR7-BV421, CD45RA-AF700, CD71-APC-H7 (BD), CD4-APC-Fire750, CD8-BV510 (BioLegend) for 30 min in the dark at 4°C, followed by fixation and permeabilization. After permeabilization, cells were stained with ki67-FITC, Granzyme B-AF700, T-bet-BV421, BAX-FITC, Bcl2-PE (BD Biosciences), Eomes-PE-Cy7 (Ebioscience), perforin-APC (BioLegend) antibodies for 30 min in the dark at room temperature. Following staining, cells were washed and acquired on an LSRFortessa.

Isolated lymphoma cells (5–10 ×10⁵ cells) were first fixed with Lyse-Fix buffer (BD558049, BD Biosciences) at 37°C for 10 min. Fixed cells were washed and re-suspended in 100 μl of FACS staining buffer (1% BSA and 2 mM EDTA in HBSS) followed by permeabilization with Perm/Wash buffer (BD557885, BD Biosciences) for 20 min at 4°C. Cells were then washed and further incubated with 20 ml of mouse IgG for 10 min at 4°C before staining with PE-BCL-2 (BD556535, BD Biosciences) or APC-BIM (supplied by AbbVie). BCL-2 and BIM expression (molecules/cell) were determined via Antibody Binding Capacity of the cells using Quantum Simply Cellular anti-Mouse IgG beads (815) and Molecules of Equivalent Soluble Fluorochrome (MESF) using Quantum APC MESF beads (823) beads (Bangs Laboratories, Fishers, IN), respectively. The standard curves were created in QuickCal^® software following instruction of Bangs Laboratories [10 ].