Guava pca

The expression of GRP78 on cell surfaces was evaluated by flow cytometry. HL-7702, DLD1 and HepG2 cells were collected using cell scrapers after double washes in PBS, and then cells were pipetted dozens to make single-cell suspension. After centrifugation at 300 g for 5 min at room temperature, cells were incubated with anti-GRP78 primary antibodies and sequentially phycoerythrin-conjugated secondary antibodies. Each of antibodies was incubated at 37 °C for 1 h in dark, and gently mixed cells every 10 min during this procedure. Cells were washed triple and loaded onto a Guava PCA flow cytometer (Millipore, USA) to measure, at least 20, 000 cells were counted per sample, and data were analyzed and exported by CytoSoft 6.0.2.
The apoptosis of cells with GBP-SubA treatment was evaluated by flow cytometry. HL-7702, DLD1 and HepG2 cells were added to 60-mm culture plates at a density of 10⁶ cells per dish and incubated overnight at 37 °C in 5 % CO₂. The purified 0.1 μg/mL GBP-SubA was added to each dish and incubated for 48 h. In the GRP78-blocking assay, the antibody of GRP78 was add in the cell medium to incubate for 1 h, and then added GBP-SubA. At last, the cells were harvested and operated according the instruction of Guava Nexin Reagent.

Cell apoptosis was determined by Annexin-V and 7-aminoactinomycin (7-AAD) double staining. Cells were seeded at a density of 2 × 10⁵/well followed by treatments as indicated. Suspended and attached cells were harvested with trypsin and centrifuged (1200 rpm) at 4 °C for 2 min. Cells were resuspended in medium to make the concentration equal to 1 × 10⁶ cells/mL. One hundred microliters of each cell suspension were incubated with 100 μL of Guava Nexin Reagent (Millipore) at room temperature in the dark for 20 min and subjected to the Guava PCA flow cytometer (Millipore). Apoptotic cells were determined with FlowJo software (version 7.6; LLC, USA).