Apc conjugated anti cd44 clone g44 26

HEK cells were grown as spheres for 9 days under Gal-1 or FKBP12 level manipulation. Spheres were stained with APC-conjugated anti-CD44 (clone G44-26, BD Biosciences) antibody and PE-conjugated anti-CD24 antibody (clone ML5, BD Biosciences) following the manufacturer's instructions. Briefly, spheres were washed with phosphate-buffered saline and incubated in 0.05% trypsin/0.025% EDTA. Cells were next washed with phosphate-buffered saline containing 2% fetal calf serum and 0.1% sodium azide (FACS buffer 1) and resuspended in the FACS buffer 1 at 10⁶ cells/100 μl. Fluorochrome-conjugated monoclonal antibodies were added to the cell suspensions at concentrations recommended by the manufacturer and incubated at 4°C in the dark for 30–40 min. After staining, cells were washed with phosphate-buffered saline containing 0.1% sodium azide (FACS buffer 2), fixed using 4% PFA and cytometric analysis was performed in a FACS LSR II (BD Biosciences) cytometer in accordance with the manufacturer's protocols. In brief, unstained cells, CD44, CD24 and double-stained control cells were used to mark the four quadrants in a dot-plot for unstained, CD44+, CD24+ and double-positive populations. The change in percentage of CD44+/CD24− cells (top left quadrant) was measured in control and samples.