Apc mouse igg2b κ isotype control

PE mouse anti-human CD24, APC mouse anti-human CD44, PE mouse IgG2a κ Isotypecontrol and APC mouse IgG2b κ Isotype Control were purchased from BD Pharmingen (San Diego, CA). Cells were resuspended at 1×10⁶cells per 100 μl of sorting buffer (1×PBS containing 0.5% bovine serum albumin) and incubated with pre-conjugated CD44-APC and CD24-PE primary antibodies for 10 min at 4°C. Three control groups were established for the first sorting: (1) cells labeled with the isotype antibodies of the above two antibodies, (2) cells labeled with the anti-CD44-APC antibody and the isotype control antibody for CD24, and (3) cells labeled with the anti-CD24-PE antibody and the isotype control antibody for CD44. The cells were washed in 1×PBS and centrifuged at 800g for 2 min. For flow cytometric analysis, cells were resuspended in sorting buffer after incubation with the primary antibodies.

Cultured cells were detached with accutase solution (Sigma-Aldrich, Inc., St. Louis, MO, USA) and washed in phosphate-buffered saline (PBS) containing 0.5% FBS. Single cells were stained for 20 min on ice in the dark, washed twice in PBS containing 0.5% FBS, and fixed in 2% paraformaldehyde. Flow cytometry analysis was performed using a FACSCalibur system (BS Biosciences, San Jose, CA), and cell sorting was performed using FACSAria II (BD Immunocytochemistry System, Franklin Lakes, NJ). Antibodies against CD24 (anti-CD24-PE, BD), CD44 (anti-CD44-APC, BD), and ESA (anti-ESA-FITC, BD) were used. FITC-mouse IgG2b, κ isotype control (BD), rat IgG1 κ isotype control FITC (eBioscience), PE-mouse IgG2a, κ isotype control (BD), and APC-mouse IgG2b, κ isotype control (BD) were used as controls.

CD44-APC Mouse Anti-Human CD44 antibody (Clone G44-26, BD Pharmingen™, Heidelberg, Germany) and APC Mouse IgG2b κ Isotype Control (Clone 27-35, BD Pharmingen™) were added to a final dilution of 1:10 to PBS-washed cells. The concentration of cells was adjusted to a range from 1 × 10⁶ to 1 × 10⁷ cells per 100 µL PBS before adding the antibodies. The suspension was incubated for 45 min at 4 °C in the dark. After incubation, the cells were washed twice with PBS by repeating centrifugation at 300× g for 8 min and adding PBS. The cells were suspended in 500 µL PBS for flow cytometry cell sorting (FACS) analysis.
Cells tagged with the CD44-APC antibody were FACS sorted. The cell suspensions were filtered first to obtain single cells for FACS. Each FACS analysis was performed by gating for single cells, then for living cells via DAPI labeling. Then, the cells were sterile filtered through a 20 μm sieve for the collection of unsorted, CD44-low and CD44-high populations directly into PBS for the subsequent experiments.