Apoptosis assay kit

Cell viability was determined by CellTiter-Glo^® luminescent cell viability assay according to the manufacturer’s protocol. Briefly, after transfection procedure, the 96-well plate and its contents were equilibrated at room temperature for 30 minutes. CellTiter-Glo^® reagent was thawed and 100 µl of the CellTiter-Glo^® reagent were added to 100 µl of medium containing cells in each well. The plate was shaken on a microplate shaker at 600 rpm for 2 minutes to induce cell lysis. The plate was then incubated at room temperature for 10 minutes. 100 µl sample of each well were transferred into a luminometer-compatible multiwell plate and luminescence of the sample was recorded by a luminometer (MicroLumat Plus LB 96 V Luminometer, Bad Wildbach, Germany).
Apoptosis assay was performed by using the apoptosis assay kit (ThermoFisher). Briefly, after transfection procedure, cells were collected and washed with PBS twice. Subsequently, cells were resuspended with 100 µl of binding buffer. 5 µl of Annexin-V-FITC solution and 5 µl of PI solution were added into the cell suspension. After incubation at room temperature for 10 minutes, 400 µl of binding buffer were added into the cell suspension and 10,000 events were analyzed from each sample by the BD FACSCalibur^®. The apoptosis and necrosis were calculated using CellQuest Pro.

Cell apoptosis assay was performed using Annexin-V-FITC/PI (propidium iodide/fluorescein isothiocyanate) Apoptosis Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instruction. In brief, cells were resuspended in binding buffer. Cells were then incubated with 5 μl Annexin-V-FITC reagent and 5 μl PI in the dark for 15 min. The samples were analyzed by flow cytometry with BD FACSCalibur (BD Biosciences, San Jose, CA, USA).

Apoptosis assay was performed using apoptosis assay kit (Invitrogen, USA). Briefly, HeLa, MCF-7 and RWPE-1 cells were seeded in 24 well tissue culture plates (about 2 × 10⁵ cells/well) and incubated for 24 h. Subsequently, culture medium was replaced with fresh medium containing 2.5 μM of LS10 along with a negative control (cells without peptide). Apoptosis induced by the peptide was examined by Annexin V/PI staining followed by flow cytometry analysis of stained cells. Briefly, after 2 h and 24 h of incubation with LS10, cells were harvested, washed twice with cold PBS and resuspended in 1X Annexin binding buffer. Cells were then transferred to fresh 1.5 ml tube containing 100 μl of binding buffer and 5 μl of FITC-conjugated Annexin V and 1 μl of PI were added. The cells were vortexed gently and incubated for 15 min at room temperature in dark. After incubation, 200 μl of 1X Annexin binding buffer was added to each tube and stained cells were analyzed by Accuri flow cytometry (BD Biosciences, USA) with CellQuest software (BD Biosciences, USA) used for analysis of the results.

The apoptosis assay kit (Invitrogen, USA) was used for cell apoptosis analysis. In 24 well culture plates, cells were seeded (about 5*10⁵ cells/well) and then incubated for 24 h. The cells were then given 100mg/ml ABGE or culture media (negative control). Apoptosis was then examined by Annexin V/PI staining, followed by flow cytometry analysis of stained cells. ABGE-treated cells were harvested after 48 hours, washed twice with cold PBS, and resuspended in 1X Annexin binding buffer. 6ul FITC-conjugated Annexin V and 1ul PI were added to a fresh 1.5 ml tube containing 100 ml of binding buffer and cells. In each tube, 200 ml of 1X Annexin binding buffer was added after 15 minutes of incubation. The stained cells were analyzed by flow cytometry (BD Biosciences, USA) with flowjo software (v10.6.2, Treestar, USA).

Phosphatidylserine translocation to the outer leaflet of the plasma membrane was detected using annexin V staining of the transfected cells. The assay was performed using the Apoptosis Assay Kit (Invitrogen, USA) according to the manufacturer’s protocol. First, the transfected DF-1 cells were harvested by trypsinization at 48 h, washed with PBS and resuspended in 1X annexin-binding buffer to a density of 1 × 10⁶ cells/mL. The cells were then labeled with 5 μL of Alexa Fluor^® 488 annexin V and 1 μL of the 100 μg/mL PI working solution and incubated at room temperature for 15 min in the dark. Thereafter, 400 μL of 1X annexin-binding buffer was added, and the cells were kept on ice and analyzed by flow cytometry (BD LSRFortessa, USA).

Apoptosis was measured using Annexin V labeling using the Apoptosis Assay Kit from Invitrogen, and the number of apoptotic cells after 24 h of DMAPT treatment was measured by flow cytometry. Both floating and adherent cells were collected and stained with Alexa Fluor-488-conjugated Annexin V and propidium iodide. Annexin V-positive cells are apoptotic, propidium iodide-positive cells are necrotic and double-positive cells are necroapoptotic.