Based on protocol by CMF; adapted by JAR 2010
% CD44+/CD24–/EpCAM+ FLOW CYTOMETRY ASSAY PROTOCOL
- Prepare FACS Buffer (PBS + 3% CD-FBS).
- Pool supernatants into labeled tubes to account for viable, non-adherent cells.
- Wash adherent cells 1X in PBS.
- Trypsinize adherent cells and pool cell suspension with pooled supernatant.
- 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.
- Resuspend cells at 106 cells/ml in FACS Buffer. Place cells on ice at this stage of protocol if a break is needed.
- Add 100 µl of 106 cells/ml suspension to a labeled FACS tube.
- 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: ________________________________________________________
- Add antibodies in the following order at the precise amount of antibody per cells stained:
CD24-PE, 4 µl per 100,000 cells (from BD Biosciences cat #555428)
CD44-APC, 4 µl per 100,000 cells (from BD Biosciences cat #559942)
ESA-FITC, 2 µl per 100,000 cells (from Stem Cell Technologies, cat #10109)
- 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: _____________________________________________________________________
- Stain cells for 15 min at RT.
- Wash cells with 1 ml FACS Buffer.
- Spin down cells at 1000 rpm for 5 min.
- Resuspend cells in 250 µl FACS Buffer.