Streptavidin coated microbeads

4% paraformaldehyde-fixed cells were centrifuged (800g × 2 min), then resuspended at 5 million cells/ml in phosphate-buffered saline (PBS, Thermo Fisher Scientific) supplemented with 5% FBS to minimize nonspecific antibody binding and cell clumping. Mouse anti-human CD44 antibody (Cat. 555476BD; Bioscience, San Jose, CA) was added to the cell suspension at a 1:500 dilution and incubated at room temperature on a shaker for 30 min. Cells were pelleted (800g × 2 min) and washed once in PBS + 5% FBS. Cells were resuspended to 5 million cells/ml, and biotinylated goat anti-mouse IgG antibody (Cat. BA-9200; Vector labs, Burlingame, CA) was added at an optimized 1:500 dilution followed by a 30-min incubation. Cells were washed in PBS + 5% FBS twice, then resuspended to 2 million/ml. High iron, 0.4 μm, streptavidin-coated microbeads (Cat. SVM-05–5H; Spherotech, Lake Forest, IL,) were then added in a cell-number dependent fashion to yield greater than 1,200 beads/cell (210 μl beads at a concentration of 5.74 × 10¹⁰ particles/ml) per 10 million cells and incubated for 30 min on a shaker at room temperature. The labeled cells were then filtered through a 70 μm nylon mesh (Thermo Fisher Scientific) to remove clumps before pipetting on top of a Percoll gradient (Thermo Fisher Scientific) for density separation.