4.11.3. Confirmation of CCK-2R Membrane Expression by Flow Cytometry

Cells of the selected line (A549) were detached with 5 mM EDTA then stained with 100 µL of phosphate buffer saline (PBS) containing 0.1% Live–Dead Fixable Dead Cell Stain near-IR-fluorescent reactive dye (Molecular Probes) on ice for 30 min to exclude dead cells from the analysis. After washing with PBS added to 1% heat-inactivated FBS (PBS/FBS), cells were incubated on ice for 30 min with 50 µL of PBS/FBS containing 20 µg/mL of anti-CCK-2R-phycoerythrin antibodies (clone E-3, sc-166690, Santa Cruz Biotechnology), 40 µg/mL of anti-CCK-2R-Dy488 antibodies (LS-C756504, LSBio), or 40 µg/mL of CCK-8 peptide (Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe) labelled with FAM supplied by GenScript Biotech (Piscataway, New Jersey, USA). Isotype control antibodies labelled with phycoerythrin (20 µg/mL, Santa Cruz Biotechnology) were used to check nonspecific binding. After washing with PBS/FBS, cells were analyzed with the FACSCantoII flow cytometer (BD).
