Annexin 5

Flow cytometry was performed to identify apoptotic and necrotic profiles. A total of 20 × 10³ cells per condition were gated and evaluated using a FACScalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and a Cell Quest Pro Acquisition software (version 5.1, BD Biosciences). Using the fluorescent probes Annexin V (Immunostep, Salamanca, Spain) and propidium iodide (PI), four populations were identified: live cells (negative for both Annexin V and PI); early apoptotic cells (positive for Annexin V and negative for PI); late apoptotic cells (positive for both Annexin V and PI); and dead cells (only positive for PI). Cells were detached by trypsinization and centrifuged (550× g, 6 min), 106/mL cells were resuspended in Annexin V binding buffer (10 mM Hepes/NaOH (pH 7.4), 140 mM NaCl, 2.5 mM CaCl₂), and they were incubated with Annexin V (50 µL/mL) and PI (2.5 µg/mL) for 15 min at RT, protected from light. Cells incubated overnight with 300 µM hydrogen peroxide (H₂O₂; Sigma-Aldrich) were used as a positive control for cell death.

Annexin V measurements were done following the manufacturer instructions: Cells from the supernatant (dead cells) were centrifuged and resuspended in Buffer 1x (Immunostep, 556454, Salamanca, Spain) Annexin V (Immunostep, 556454) was added at 5 µL/sample, and incubated for 15 min at 37 °C + 5% CO₂ in darkness; DAPI was subsequently added. BioVision kits were used for caspase 8 (#K188-25; Madrid, Spain) and caspase 9 (#K189-25) in vivo activity measurements [58 (link),63 (link)]. As negative controls, z-VAD-FMK was added for every experimental condition, following the instructions of the supplier.

Raji cell line, which was provided by the existing collection of the Instituto de Salud Carlos III (Madrid, Spain), was used as a target to determine the antibody-dependent cellular cytotoxicity (ADCC) capacity of PBMCs isolated from CML individuals and healthy donors, as described previously [20 (link)]. Briefly, Raji cells were labeled with PKH67 green fluorescent cell linker (Merck KGaA, Darmstadt, Germany) and then coated with rituximab (50 μg/mL) (Selleckhem, Houston, TX, USA) for 4 h. Labeled rituximab-coated Raji cells were then co-cultured for 18 h with PBMCs (1:2 ratio). Early apoptosis of Raji cells was evaluated via staining with Annexin V conjugated with phycoerythrin (PE) (Immunostep, Salamanca, Spain). Data acquisition was performed with a BD LSRFortessa X-20 flow cytometer and data analyses were performed with FlowJo software v10 (Tree Star Inc.).