Ab14085

All cell groups were seeded into a 6-well plate at a density of 10 × 10⁵ cells/well. Following a 16-h period, the culture medium was replaced by a fresh culture medium containing different concentrations of cisplatin (0, 0.5, 1, 2, 3 μg/mL), with five identical wells each being set at a different concentration. Following 48 h of drug treatment, the cells were centrifuged and re-suspended in 200 μL of binding buffer solution. After that, 10 μL of Annexin V-FITC (ab14085, Abcam Inc, Cambridge, MA, USA) and 5 μL of propidium iodide (PI) were gently mixed and underwent a 15-min period of incubation, while avoiding light. Afterwards, cells were added with 300 μL of binding buffer solution, and the cell apoptosis was detected via FCM at an excitation wavelength of 488 nm.

AnnexinV-FITC/PI staining (Abcam, ab14085) and analysis by flow cytometry was used to assess viability in the primary cells incubated with rhEPO in the presence or absence of anti-EPOR antibodies (Santa Cruz: sc101444, clone MM0031-6G7). The anti-EPOR was preincubated for 30 min together with the primary cells before rhEPO was added. Cell proliferation assay was performed after 48 h incubation with rhEPO using CellTiter-Glo® Luminescent Cell Viability Assay (Promega, Madison, Wisconsin) according to the manufacturer’s instructions.

Neuronal cells were cultured in a 5% CO₂ incubator at 37°C for 48 h. The cells were collected and resuspended in 200 μL binding buffer after centrifugation. The cells were then mixed with 10 μL Annexin V-FITC (ab14085, Abcam, Cambridge, UK) and 5 μL propidium iodine (PI) for 15 min avoiding exposure to light, and then 300 μL of binding buffer was added. Apoptotic cells were detected using a flow cytometry (Becton, Dickinson and Company, Franklin Lakes, NJ, USA).

Annexin V–FITC assay was used to detect apoptosis. The transfected cells were seeded in six-well plates at a density of 1 × 10⁵ per well 48 h post-transfection. After washing with PBS, 10 μL of Annexin V–FITC (ab14085, Abcam, Cambridge, MA, USA) and 5 μL of propidium iodide were added to each well and mixed gently. After 15-minutes, 300 μL of the binding buffer was added and cell apoptosis was detected using FACSCalibur^TM (BD Biosciences, San Jose, CA, USA).

P. falciparum strain 3D7 cells were cultured in RPMI 1640 medium supplemented with 25 mM HEPES, 0.5% AlbuMAX II, 1.77 mM sodium bicarbonate, 100 μM hypoxanthine and 12.5 μg ml⁻¹ gentamicin sulfate at 37°C. Parasites were maintained at 1 to 1.5% hematocrit and 5% parasitemia. Fresh O⁺ human RBCs were isolated from healthy human donors. Parasites were diluted after every 2 days by splitting the flask into 2 to 3 flasks in order to maintain the parasitemia around 5%. Parasites were synchronized at the schizont stage using a 63% Percoll density gradient and synchronized at the early ring stage using 5% sorbitol. For the stress experiment, parasites were kept at 8% parasitemia and 2% hematocrit. Parasite growth was monitored using Giemsa staining of a thin blood smear. Parasites were maintained in an incubator with 5% CO₂. Annexin V-FITC (ab14085; Abcam) staining was performed as mentioned in the protocol.

After transfection for 48 h, cells were collected and dispersed into cell suspension with 0.25% trypsin, 1 × 10⁶ cells/mL. As for cell cycle detection, 100 µL cell suspension was incubated with 50 µL PI dye containing RNAase avoiding light exposure for 30 min. Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining was conducted for apoptosis assessment. In short, the cells were stained with 10 µL Annexin V-FITC (ab14085, Abcam, Inc., Cambridge) and 5 µL PI for 15 min devoid of light exposure. The cell cycle and apoptosis were assessed on a flow cytometer (BD Biosciences, FL, Lakes, NJ) at an exciting wavelength of 488 nm.