Bd555478

For FACS, cells were dissociated using Accutase and resuspended in PBS containing 0.5% BSA. The cells were stained with FITC-conjugated CD44 (BD555478) or isotype control antibody (BD555742) from BD Biosciences on ice for 30 min. Cells were then washed with PBS and analyzed on a BD FACSCalibur (BD Biosciences, San Jose, CA) using Cell Quest software.
CD44-positive cells were sorted by a magnetic-activated cell sorting (MACS) system (Miltenyi Biotech, San Diego, CA). After collecting spheroids, cells were washed with phosphate-buffered saline (PBS), dissociated to single cells using Accutase, and stained with CD44-Micro Beads on ice for 30 min. Cells were then passed through a LS magnetic column where CD44-positive cells were retained. The CD44-positive cells were then eluted from the column after removal from the magnet. Quantitative analysis of CD44-positive cells was performed by immunofluorescence using FITC-conjugated CD44 antibody.

Cells were dissociated using Accutase and resuspended in PBS containing 0.5% BSA. The cells were stained with FITC-conjugated CD44 (BD555478) or isotype control antibody (BD555742) from BD Biosciences on ice for 30 min. Cells were then washed with PBS and analyzed on a BD FACSCalibur (BD Biosciences) using Cell Quest software.

For FACS, cells were dissociated using Accutase (Innovative Cell Technologies) and resuspended in PBS containing 0.5% BSA. Cells were stained with FITC-conjugated anti-CD44 (BD555478) or isotype control antibody (BD555742) and analyzed on a FACSCalibur (BD Biosciences) using Cell Quest software. CD44-positive cells were collected using a magnetic cell-sorting system (Miltenyi Biotech). Briefly, cells were dissociated using Accutase, stained with CD44-Micro Beads, and passed through a LS magnetic column that retains CD44-positive cells. CD44-positive cells were then eluted from the column after removal of the magnet and quantified by immunofluorescence using FITC-conjugated CD44 antibody (555478; BD Biosciences).