Apoptosis detection kit

Cellular integrity was validated by propidium iodide (PI) staining using an apoptosis detection kit (BioVision, USA). In brief, 1 × 10⁵ cells were grown on poly L-lysine coated coverslips kept in six-well plates for 24 h. Cells were subjected to treatments as described in the figure legends. Post incubations cells were stained with propidium iodide and images were captured under a fluorescent microscope (DP71, Olympus, Japan) in DIC and fluorescent mode, the result represents merged images of cells in DIC with PI stained nuclei. More than 200 cells from three different fields were analyzed.

HTR-8/SVneo cells (1 × 10⁶ cells per well) were plated onto 6-well culture plates. After 48 h of sPIF treatment, cells were stained using an apoptosis detection kit (Abcam), according to the manufacturer's instructions. Flow cytometry analysis of annexin V-FITC staining was performed using an imaging flow cytometer (Amnis/EMD, Imagestream, Merck-Millipore, Darmstadt, Germany). A total of 1000 events were acquired at × 40 magnification, and annexin V-positive cells were quantified using IDEAS 5.0 software (Amnis/EMD, Darmstadt, Germany). Bright field images were used to quantify and verify cell integrity. The apoptosis rate was determined by normalizing annexin V-positive cells against the total number of cells. Etoposide (42 μM) was used as positive control (data not shown).

Normal human lung fibroblast cells (IMR-90) were obtained from ATCC #CCL-186. Cells were maintained in DMEM (Dulbecco’s Modified Eagle Medium) supplemented with 10% fetal calf serum (FCS, Hyclone) containing 1% penicillin-streptomycin (Invitrogen) and maintained in a humid atmosphere at ${37}^{\circ }\text{C}$ containing 5% ${\text{CO}}_2$ . When cells were grown to 70% confluence, they were subcultured at $\frac{1}{5}$ dilution for later passaging.
The apoptotic assay of IMR90 was conducted by Apoptosis Detection Kit (ab214663, Abcam) according to the manufacturer’s protocol. Briefly, cells were detached using 0.05% trypsin and washed twice with PBS. Then, samples were resuspended in 1x annexin-binding buffer and incubated with $5\mu \text{L}$ Annexin V-FITC and $5\mu \text{L}$ 7-amino-actinomycin D (7-AAD) for 15 min at 37 ${}^{\circ }$ C, avoiding light. Finally, events were acquired with a Guava Easycyte Flow cytometer (Millipore-Sigma) and analysed by Freecyto and Flowjo software individually to quantify the distribution of cells.

Induction of cell death after Coleon U treatment was assessed by Abcam Apoptosis Detection Kit through dual staining with Annexin V—FITC/Propidium Iodide (AV/PI) according to the manufacturer’s instructions. NCI-H460 and NCI-H460/R cells were seeded in adherent 6-well plates at a density of 50,000 cells/well and incubated overnight. Both cell lines were treated with Coleon U (15, and 30 µM). Treatment with 500 nM PTX was used as a positive control in both cell lines. After 72 h, both attached and floating cells were collected and washed in PBS. After centrifugation, cells were resuspended in 100 µL of binding buffer containing AV and PI (1:1 ratio), and incubated for 15 min at room temperature in the dark. Cells were then analyzed on a CytoFLEX flow cytometer (Beckman Coulter, Indianapolis, IN, USA). The fluorescence intensities of AV and PI were measured in green fluorescence channel 1 (FL1) at 525 nm and red fluorescence channel 2 (FL2) at 585 nm, respectively. In each sample, the acquisition of 20,000 cells was performed, and the percentages of viable (AV− PI−), early apoptotic (AV+ PI−), late apoptotic (AV+ PI+), and necrotic (AV− PI+) cells were analyzed by CytExpert 2.4 software (Beckman Coulter, Indianapolis, IN, USA).

5×10⁶ PC12 cells were labeled with annexin V-FITC and propidium iodide (PI) (Apoptosis Detection Kit, abcam, Cambridge, England). Cells were analyzed using a dual-laser Accuri C6 flow cytometer (BD, Heidelberg, Germany). Annexin V-FITC and PI signals were excited using a 488-nm laser light. For dual labeling, fluorescence emissions of individual fluorophores were corrected for spectral overlay using electronic compensation.