Annexin 5 fluorescein isothiocyanate propidium iodide

Annexin V-fluorescein isothiocyanate/propidium iodide dual staining was employed to distinguish early and late apoptotic cells (BD Pharmingen, San Jose, CA, USA). Fluorescein isothiocyanate -conjugated Annexin V was used to quantify the loss of asymmetry of phosphatidylserine on cell membranes involved in apoptosis while propidium iodide differentiates the early apoptotic, the late apoptotic and necrotic cells [22 (link)]. Briefly, HT29 cells (3 × 10⁵ cells/well) were seeded in a 6-well plate and treated with the orientin for 48 h. Cells were gently washed twice with phosphate buffered saline accompanied by tryptic digestion (0.25% trypsin/EDTA) and washed yet again with PBS. All the cells including the floating and adherent ones were harvested, pooled and incubated with dual stain for 15 min on ice under dark. The cells were counted by a flow cytometer (Becton-Dickinson, San Jose, CA, USA).

Annexin V-fluorescein isothiocyanate/propidium iodide staining was performed using a kit from BD Pharmingen (San Jose, CA, USA).^{43 (link)} Briefly, cells were detached by trypsinization and collected by centrifugation at 1000 × g for 5 min. The cells were then resuspended in binding buffer at a density of 1–2 × 10⁶ cells/ml. The 100 μl single-cell suspension (1–2 × 10⁵ cells) was incubated with 5 μl Annexin V-fluorescein isothiocyanate and 5 μl propidium iodide for 15 min at room temperature. Finally, the mixture was diluted with 500 μl binding buffer and analyzed with a FACSCalibur flow cytometer (NovoCyte, ACEA Biosciences, San Diego, CA, USA). For each sample, total of 10 000 events were counted.

To obtain activated T cells, human PBMCs (StemEry Biotech, Fuzhou, China) were treated with a T-Cell Activation/Expansion Kit (130-091-441, Miltenyi Biotec, Bergisch Gladbach, Germany) for 24 h following the manufacturer’s instruction. At the same time, RKO cells treated with the TRAPPC4 siRNA or control siRNA were seeded into a 96-well plate at a density of 3000 cells per well. Then, RKO cells were then co-cultured with activated PBMC cells at a ratio of 1:10 in the 37°C incubators for 6 h. To detect apoptotic cells, all the cells were harvested with trypsin into different centrifuge tubes and incubated with APC-conjugated anti-human CD45 antibodies (BD Biosciences) in staining buffer at 4°C for 30 min in the dark. The cells were then washed twice with cold PBS and stained with Annexin V fluorescein isothiocyanate/propidium iodide (BD Biosciences) following the manufacturer’s protocol to detect apoptotic cells. Apoptosis of the CD45⁻ cells (tumor cells) was further examined using flow cytometry.