Annexin 5

Short-term cell death was determined with an Incucyte FLR or Zoom imaging system (Essen Bioscience)⁵⁸ . Cells were treated as indicated in the Figure legend together with 30 nM Sytox green and imaged every 1 or 2 h. Analysis was performed using the Incucyte software and the number of dead (Sytox green positive) cells was normalised to the confluency at t = 0. Alternatively, cells were pre-treated for 1 h with 50 nM Syto 21, followed by cytotoxic treatments as indicated together with 5 μg/ml propidium iodide. Long-term colony formation assay was performed by plating 1000 cells per 6 well and treatment as described in the Figure legend. After 48 h of treatment with supernatant, the medium was changed to 2.5 μM venetoclax/S63845. Medium was changed to regular growth medium after an additional 48 h, and resulting colonies were stained with crystal violet after an additional 5 days. Cell death analysis via FACS was performed using Annexin V - propidium iodide staining^{59 (link)}. In short, treated cells were harvested and stained with 5 μg/ml propidium iodide and Annexin V (Biolegend) in Annexin V-binding buffer (10 mM Hepes pH 7.4, 140 mM NaCl, 2.5 mM CaCl₂) for 15 min. Flow cytometry was conducted on a BD FACSCalibur machine with BD CellQuest software and analysed using Flowing software; cells negative for propidium iodide and Annexin V were considered alive.

Adherent cells were trypsinised and prepared as single cell suspension in U-bottom 96-well plates as described in “Detailed analysis of cell cycle distribution”. Annexin V + PI solution was added to the cell suspension to make up final concentrations of 5 μL/mL of Annexin V (BioLegend, FITC), 200 μg/mL RNAse A (Sigma-Aldrich), and 50 μg/mL PI solution (Sigma-Aldrich) in 1 × PBS. PI + cells denoted late apoptotic/necrotic cells, PI − Annexin V + cells early apoptotic, and PI − Annexin V-cells live cell populations. Cells were analysed via FACS Attune (Thermo Fisher) BL1 (Annexin V), YL2 (PI).

Human DLBCL cells (HBL1, MD-901, U2932 and SUDHL4) transduced with pTRIPZ-miR-28 or pTRIPZ-scramble were counted, and 200,000 cells were seeded in duplicate on a 48-well plate in fresh medium containing doxycycline at 0.5 μg/ml to induce miRNA expression. Serial dilutions of ibrutinib (Selleck Chem) were prepared in vehicle (DMSO), and equal volumes of the diluted drug were added to cells to achieve the desired final concentration. At least two independent experiments were performed per cell line. The number of viable cells was determined 72 h after the start of ibrutinib treatment using CellTiter-Glo® reagent. Luminescence was measured with an Orion Plate Luminometer reader (Berthold). Luminescence values in medium-only wells were subtracted to yield relative luminescence units (RLU), and the data were normalized to DMSO-treated or ibrutinib-treated control cells. The percentage of apoptotic (Annexin V⁺ Dapi⁻) or dead (Dapi⁺) cells was measured 72 h after ibrutinib treatment by Annexin V (Biolegend, Cat#640932) and Dapi staining, and RFP expression was measured by flow cytometry in an LSRFortessa High-Parameter Flow Cytometer and analyzed with FlowJo V10.4.2 software.

PBMCs from PROMOTE subjects (28 months of age; no chemoprevention control arm) were thawed and rested overnight either in standard media (untreated), 5uM camptothecin (Sigma) or P. falciparum schizont extract (PfSE) or protein extract from uninfected RBCs (uE) at an effector:target ratio of 3:1. To test for induction of apoptosis, stains for AnnexinV (Biolegend) or YoPro (Invitrogen), or activated Caspase 3 FITC (BD) were used according to the manufacturer’s instructions in combination with the following antibodies: AnnexinV and YoPro staining—CD3 (OKT3) Brilliant Violet 650, CD4 (RPA-T4) APC-Cy7, CD127 (A019D5) APC, CD25 (BC96) PE-Cy7 from Biolegend; for Caspase3—CD3 (OKT3) Brilliant Violet 650, CD4 (RPA-T4) PerCP, CD127 (A019D5) Pacific Blue, CD25 (BC96) PE-Cy7. T_regs were gated as CD3+CD4+CD25+CD127^dim. Sensitivity to apoptosis was measured ex vivo (untreated control), after induction with camptothecin (fold change compared to untreated), and after stimulation with P. falciparum schizont extract (fold change comparing PfSE to uE).

Mouse activated T cells were cultured in the absence or presence of DEX (1 × 10⁻⁷ M). at different time points (6, 12, 24, and 48 h), percentage of apoptosis was determined by using FITC-labelled Annexin V and propidium iodide (PI) (Biolegend). Briefly, 1 × 10⁶ cells were resuspended in 100 μL of binding buffer (Biolegend) containing Annexin V and PI according to manufacturer’s instructions. After incubation for 15 min in the dark at room temperature, cells were analyzed with a Beckman Counter FC500 using FlowJo software.

Thymocytes were harvested from young (2 months old) and aged (16 months old) Rag-GFP reporter mice, and stained with surface CD markers as described above. Then, the cells were washed with Annexin-V binding buffer and incubated in APC-Annexin-V (BioLegend, Cat# 640920) at a 1:20 dilution with Annexin-V binding buffer (10 mM Hepes adjusted to pH 7.4, 140 mM NaCl and 2.5 mM CaCl₂) for 15 min at room temperature in the dark. The cells were then washed with PBS and fixed and permeabilized with fixation/permeabilization buffer, followed by eBioScience intracellular staining protocol for FoxP3 and GFP. Positive control cells were aliquoted from the thymocytes and made by incubating at 55°C for 20 min to induce cell death before staining.