Apc conjugated anti cd9 clone h19a

Cells were grown overnight on poly-d-lysine–coated coverglasses in CM and then transferred to prechilled 4°C CM. Cells were washed in 4°C phosphate-buffered saline (PBS) and then incubated for 30 min at 4°C with prechilled 4°C PBS (200 μl) containing 4 μl of fluorescein isothiocyanate (FITC)–conjugated anti-CD63 (clone H5C6, BioLegend) and 4 μl of allophycocyanin (APC)–conjugated anti-CD9 (clone H19a, BioLegend). Excess Ab was removed by two washes with 4°C PBS. Cells were then fixed immediately or transferred to CM at 37°C and incubated for 30 min at 37°C. Fixation was with 3.7% formaldehyde in PBS for 20 min. Cells were then incubated with DAPI to stain the nucleus and then examined by confocal fluorescence microscopy and imaged to assess the subcellular distribution of plasma membrane–labeled CD63 and CD9. Confocal fluorescence microscopy was performed using a Zeiss LSM880 microscope with gallium-arsenide phosphide (GaAsP) detectors and a 63×/1.4 NA (numerical aperture) Plan-Apochromat objective. Images were assembled into figures using ImageJ and Adobe Illustrator.

Cells were released by trypsinization (TrypLE, Thermo Fisher Scientific), and cell clumps were removed using a cell strainer (Falcon, catalog no. 352235). Approximately 500,000 cells were then concentrated by a brief spin at 400g for 5 min and resuspended in 100 μl of 4°C FACS buffer (1% FBS in PBS) containing 2 μl of FITC-conjugated anti-CD63 (clone H5C6), 2 μl of APC-conjugated anti-CD9 (clone H19a), 2 μl of phycoerythrin (PE)–conjugated anti-CD81 (clone 5A6), or 2 μl of peridinin chlorophyll protein (PerCP)–conjugated anti-Lamp1 (clone H4A3) and 2 μl of PE-conjugated anti-Lamp2 (clone H4B4), all from BioLegend, for 30 min with gentle mixing every 10 min. Cells were washed three times with 1 ml of 4°C FACS buffer, with cells recovered by 400g spin for 5 min at 4°C. After the final wash, cells were resuspended in FACS buffer with DAPI (0.5 μg/ml) and analyzed using CytoFLEX S flow cytometer (Beckman Coulter). Flow cytometry histograms were generated using FlowJo (v10.8.1). For separation by FACS, labeled cells were sorted into single cells in a 96-well plate, on the basis of high or low cell surface labeling for CD63 or CD9.