Annexin 5 fluos staining kit

Apoptosis was measured using the Annexin-V-FLUOS Staining Kit (Sigma-Aldrich) as recommended by the manufacturer's instructions. Briefly, after 24 h of incubation with Ac5sGlcNAc or ThiametG, cells were gently scraped (we did no use trypsin to detach cells to avoid the unspecific exposure of Annexin V), centrifuged and washed twice in PBS. Annexin V-FITC in the staining buffer and propidium iodide were added to cell suspension and incubated for 10 min at room temperature. Cell were washed and analyzed with Attune Flow Cytometer.

Cell death induced by apoptosis was detected by AnnexinV Apoptosis Detection Kit II (BD Pharmingen™) or Annexin-V-FLUOS Staining Kit (Sigma-Aldrich) according to manufacturers’ instructions. Cells were seeded in 6-well plates and allowed to attach for 24 h. One hour after treatment with SAR (0.125 μM, 0.25 μM), cells were irradiated with 8 Gy single dose and treated with 5-aza-dC (0.1 μM, 0.5 μM) or TMZ (50 μM, 100 μM). Cells were harvested by trypsinization 4, 24, and 96 h after IR, washed twice with PBS and stained with Annexin V FITC antibody and propidium iodide. Cell staining was measured by flow cytometry (Beckman Coulter, EPICS XL). Additionally, apoptosis-induced DNA fragmentation was determined in sub-G1 fraction of cell cycle analysis (Nicoletti assay) after propidium iodide staining. Cells were treated and harvested as mentioned above, washed twice with PBS, fixed with 70% ethanol, and stored at − 20 °C overnight. After two washes with PBS cells were incubated with 0.1 mg/ml RNAseA solution (Sigma-Aldrich) at 37 °C for 20 min. Then, 50 μg/ml propidium iodide (Sigma-Aldrich) was added and the solution was incubated at 4 °C for 10 min before DNA content was measured by flow cytometry. Sub-G1 fraction was determined using EXPO32 software (Beckman Coulter).

PBMC, Hep G2, and HEK-293 cells were treated for 1 hr with KB-151 and KB-208 at a 250 µM concentration. Thimerosal (100 µM) was utilized as a positive control for viability (diluted in RPMI). Staining with Annexin V and PI was performed according to the manufacturer’s procedure for the Annexin-V-FLUOS Staining Kit (Sigma), and samples were run through an SP6800 Spectral Cytometer.

Cells (5 × 10⁵ cells/plate) were seeded in 60 mm dishes overnight, followed by treatment with the indicated concentrations of ganetespib and/or lapatinib for 24 hours. Then, cells were collected, washed, and stained using an Annexin-V-FLUOS staining kit (Sigma-Aldrich) according to the manufacturer’s instructions. The samples were analyzed using a Guava EasyCyte 8 Flow Cytometer and the distribution of apoptotic cells was calculated using ModFit software.

To determine apoptotic rates of MeHg-treated cells, MCF7 cells were treated with MeHg as described above. On day 5, the apoptotic rates were determined with an Annexin-V-Fluos staining kit according to the manufacturer’s instructions (Sigma-Aldrich, United States). In short, the cells were incubated in Annexin-V-FITC (Sigma-Aldrich, United States) for 30 min, washed 3x with HBBS, and analyzed by immunofluorescence [29 (link)].

After corresponding treatments, CNE-2 cells (1 × 10⁴) were seeded into 6-well plates. The EdU assay was performed using the Click-iT™ EdU Cell Proliferation Kit (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. Images were taken under a fluorescence microscope (Nikon, Japan). Cell apoptosis was determined using the Annexin-V-FLUOS Staining Kit (Sigma-Aldrich, USA) according to the manufacturer’s instructions.