<p>Based on protocol by CMF; adapted by JAR 2010</p><p>% CD44+/CD24-/EpCAM+ FLOW CYTOMETRY ASSAY PROTOCOL</p><p>1. Prepare FACS Buffer (PBS + 3% CD-FBS). 2. Pool supernatants into labeled tubes to account for viable, non-adherent cells. 3. Wash adherent cells 1X in PBS. 4. Trypsinize adherent cells and pool cell suspension with pooled supernatant. 5. Count viable cells. If difficult to detect viability based on light refraction under the microscope, use a trypan blue exclusion to account for viable cells only. 6. Resuspend cells at 106 cells/ml in FACS Buffer. Place cells on ice at this stage of protocol if a break is needed. 7. Add 100 ul of 106 cells/ml suspension to a labeled FACS tube. 8. For control tubes: determine how many different culture conditions were used for the experiment, and take a representative equal volume of 106 cell/ml suspension for each condition to accommodate the control stains needed for proper compensation on the Calibur. Control tubes cell suspension: ______9. Add antibodies in the following order at the precise amount of antibody per cells stained: a) CD24-PE, 4 ul per 100,000 cells (from BD Biosciences cat # b) CD44-APC, 4 ul per 100,000 cells (from BD Biosciences cat # c) ESA-FITC, 2 ul per 100,000 cells (from Stem Cell Technologies, cat # 10. Add antibodies to the control stains. Ideally, each antibody should be stained separately, an unstained should be included, and an isotype of each antibody for the triple stain. Control stains: ______11. Stain cells for 15 min at RT. 12. Wash cells with 1 ml FACS Buffer. 13. Spin down cells at 1000 rpm for 5 min. 14. Resuspend cells in 250 ul FACS Buffer. </p><p>NOTES: - I use the same instrument and compensation settings for the same cell line with good reproducibility IF the FSC/SCC plots look good. Very rarely have I had to alter these for the same cell line within a year time period. - My general compensation settings and instrument settings for MCF7 cells are:</p>