Annexin 5 fitc pi apoptosis detection kit

After gene transfection, tumor cell viability was assessed using Cell Counting Kit-8 (CCK8) kit (Dojindo Molecular Technologies, Kumamoto, Japan) according to the manufacturer's protocol. Experiments were repeated at least three times with similar data.
Furthermore, apoptosis assay was also performed after 48 h transfection with miR-31 mimic or siRNA E2F2intoSGC-7901 and MGC-803 cells using Annexin V FITC/PI Apoptosis Detection Kit (Roche, Basel, Swiss) and Accuri C6 flow cytometer and Cell Quest Pro Software (BD Biosciences, San Jose, CA).
In addition, cell-cycle analysis were performed additional culture of 12 and 24 h after 48 h transfection with miR-31 mimic or siRNA E2F2 into SGC-7901 and MGC-803 cells using cell cycle detection kit (Keygen, Nanjing, China) and Accuri C6 flow cytometer (BD Biosciences).

PASMC proliferation was measured using the BrdU Cell Proliferation Assay Kit (Sigma-Aldrich). For BrdU incorporation assays, cells were plated in 96-well plates at a density of 5 × 10³ cells/well in culture medium with 10% FBS under standard culture conditions. After confluence > 80%, they were incubated in normoxia or 3% oxygen for 0 h, 24 h,48 h and 72 h treatment with MSC-CM or MSC-exo, and then they were incubated with 5-BrdU labeling solution for another 2 h. Detection antibody and HRP-conjugated secondary antibody were added, absorbance of BrdU plates was measured at 450 nm. PAEC apoptosis were detected using Annexin V-FITC/PI apoptosis detection kit (Roche Diagnostics, Indianapolis, IN, USA) according to the manufacturer’s instructions as our previously reported [21 (link)]. Briefly, 1 × 10⁶ cells treatment with MSC-CM or MSC-exo and then incubated in normoxia or 3% oxygen for 48 and 72 h, they were collected and suspended in 500 μl binding buffer, 5 μl Annexin V-FITC and 5 μl PI were added to each sample and incubated for 15 min in the dark. The percent of apoptosis were analysis by FACScan flow cytometry (FACS LSRFortessa; BD Biosciences, Franklin Lakes, NJ, USA). Triplicate experiment with triplicate samples were performed.

We used the Annexin V-FITC/PI Apoptosis Detection Kit (Roche, Basel, Switzerland) to detect cell apoptosis. SW620 cells were cultured overnight in 12-well plates at a density of 1 × 10⁵ per well. The cells were divided into four groups according to the treatments that they received: the blank control group, X-ray irradiation group, Cu-Cy group, Cu-Cy-PDT group. At 24 hours after PDT treatment, the cells were collected by trypsinization and washed twice in PBS. They were resuspended in 500 μl 1 × binding buffer and stained with 5 μl of Annexin V-FITC plus 5 μl of PI. Cells were incubated for 10 min at room temperature in the dark. Finally, the fluorescence was immediately analyzed using a FACScan flow cytometer (BD Biosciences, CA, USA).

Detection of apoptosis by Flow cytometry was performed using the Annexin V-FITC/PI Apoptosis Detection Kit (Roche, Switzerland). The transfected cells were harvested with trypsinization (no Ethylene Diamine Tetraacetic Acid, EDTA containing). Staining was performed according to the producer’s manual. Flow cytometry (BD, USA) was performed immediately.