Annexin 5 apoptosis detection kit

Apoptosis of the PG545 and/or gemcitabine-treated cells was determined as previously described [32 (link)]. Cells were quantitated by double staining with Annexin V-FITC and propidium iodide (PI) using the Annexin V-Apoptosis Detection kit (Biovision, USA). Apoptotic cells were analyzed by FACSCalibur (Becton Dickinson, USA) to define as those positive cells for Annexin V with or without PI staining.

Apoptotic cells were quantified using the Annexin V apoptosis detection kit (Bio-Vision, SF) according to the manufacturer's protocol. Cells were treated with cisplatin as indicated and analyzed using a FACS Canto II flow cytometer (BD Biosciences) and FlowJo software.

Apoptotic cells were quantified using the Annexin V Apoptosis Detection Kit (K129, BioVision, Milpitas, CA) and propidium iodide as previously described^{9 (link)}.
For analyzing interaction of GPR15 and TME5, EA.hy926 cells were pre-incubated with either rTM or control diluent followed by incubation with V5-tagged TME5 overnight. Cells were incubated with anti-GPR15 and V5 antibodies followed by staining with Alexa Fluor 488- and Alexa Fluor 647-conjugated secondary antibodies.
For analyzing expression of CD31 on murine ECs, cells were trypsinized followed by staining with anti-CD31 (102407, BioLegend, San Diego, CA).
Data were acquired on a BD LSRFortessa flow cytometer and analyzed by using FlowJo software.

The detection of phosphatidylserine externalization was performed using an Annexin V Apoptosis Detection Kit (K101-100 BioVision CA, USA), made up of annexin V–fluorescein isothiocyanate (AnV–FITC) and propidium iodide–phycoerythrin (PI–PE), which are able to differentiate viable from necrotic and apoptotic cells. The aliquots of experimental samples were washed with PBS (Euroclone, Milan, Italy), centrifuged, and suspended in 500 μl of Annexin binding buffer to obtain a cell count of approximately 1 × 10⁵. Five microliters of AnV–FITC and 5 μl of PI–PE (50 μg/ml) were added to each cell suspension. The samples were incubated at RT for 5 min in the dark and then analyzed by a flow cytometer. Flow cytometry analysis was performed by plotting green fluorescence (FL1)/AnV–FITC vs. red fluorescence (FL2)/PI–PE positive cells. The combination of AnV and PI allows the discrimination of four cell categories: viable cells (AnV−/PI−), early apoptotic cells (AnV+/PI−), late apoptotic cells (AnV+/PI+), and necrotic cells (AnV−/PI+). The sum of apoptotic cells was also calculated. Flow cytometry data acquisition was performed on a FACSscan Calibur (Becton Dickinson, Milan, Italy) equipped with 488- and 633-nm lasers and running CellQuest Software (Becton Dickinson, CA, USA). Ten thousand events were collected for each sample (43 (link)).

Forty-eight hours after incubation with CD47 blocking monoclonal antibody (B6H12; eBioscience) or mouse IgG1κ antibody (eBioscience), apoptosis of the gastric cancer cells was evaluated by flow cytometry using an Annexin V Apoptosis Detection Kit (BioVision, Milpitas CA) according to the manufacturer's protocol. The PI-negative Annexin V-positive cell fraction was defined as comprising apoptotic cells. This experiment was performed in triplicate.

After growing to 80–90% confluence in 6-well plates, cells were treated with cisplatin as indicated for 24 hr. Apoptotic cells were labeled using the Annexin V apoptosis detection kit (Bio-Vision, SF, USA) according to the manufacturer's instructions, and cells were analyzed using a FACS Canto II flow cytometer (BD Biosciences, USA) and Flowjo software.