Clone da1e

To determine intracellular expression levels of BH3 proteins, cells were fixated in 2% paraformaldehyde, permeabilized using perm/wash buffer (BD Bioscience, Franklin Lakes, NJ, USA) and subsequently stained with fluorescently labeled antibodies against BCL-2 (clone Bcl-2/100, BD Bioscience), BCL-XL (clone 54H11, Cell Signaling, Cambridge, UK), MCL-1 (Clone D2W9E, Cell signaling) or respective isotype controls (Cat.: 556357, BD Bioscience; clone DA1E, Cell Signaling). Dead cells were excluded by Fixable Viability Dye staining. If not otherwise stated, reagents and antibodies were purchased from eBioscience. Flow cytometric analysis was performed on a BD FACS Canto II (BD Bioscience) and data were analyzed using FlowJo software (TreeStar Inc., Ashland, OR, USA).
Annexin V staining was performed on PDX AML-388, ALL-199 and ALL-265 cells isolated from the mouse BM 72 h after TAM treatment or thawed and treated in vitro, using PE/Cy7 Annexin V (BioLegend, 640949) according to the manufacturer’s instruction and analyzed by flow cytometry (LSRII, BD Bioscience).

Activated PBMCs were stained with anti-BDCA4-APC (CD304, Neuropilin-1, BioLegend, Cat. No. 354506) for 30 min at 4 °C then were fixed and permeabilized with BD Cytofix/CytopermTM Plus Fixation/Permeabilization Kit (BD Biosciences) according to the manufacturer’s instructions. After washing cells were incubated with anti-cleaved IL-1β (Asp116; clone D3A3Z; Cat. No. 83186S) or anti-cleaved caspase-1 (Asp297; clone D57A2; Cat. No. 4199S) antibodies (all from Cell Signaling) for 30 min at 4 °C. After washing cells were incubated with PE-conjugated IgG1 secondary antibody (Cat. No. 406421; BioLegend) for 30 min at 4 °C. As an isotype control rabbit IgG (Cell Signaling, clone DA1E, Cat. No. 3900S) was used. Stained cells were dissolved in Mowiol^® 4–88 mounting media (Sigma-Aldrich, Cat. No. 81381).
For confocal microscopy 100,000 cells were pipetted on 8-well μ-Slides (chambered coverslip, ibidi GmbH). Cells were visualized immediately by a Zeiss LSM880 confocal microscope. BDCA4-APC was excited at 633 nm and IgG1-PE was excited at 543 nm. Fluorescence emission was detected through 650 to 670 nm and 560 to 615 nm band-pass filters. Images were taken in multi-track mode to prevent cross-talk. Image stacks of 1024 × 1024 pixel, 1.5 μm thick optical sections were obtained with a 40× C-Apochromat water immersion objective (NA = 1.2).