EpCAM CD24 CD49f Cell Line Flow (Click to Download)

  1. Collect cells by trypsinization to generate a single cell suspension
    1. If needed, DNase I treat and/or filter cells through a 40 µm or 20 µm mesh to ensure removal of clumps
  2. Resuspend cells at 1 x 106 cells/ml in PBS + 2% calf serum
  3. Aliquot 200,000 cells to FACS tubes for staining
  • For each experiment include the following tubes from at least one line/condition:

Unstained (to set FSC/SSC)

Isotype control

FITC-CD24 alone

PE-CD49f alone

APC-EpCAM alone (optional)

Triple stain

  1. Add antibodies to the cells in the dark, flick tube after addition of each antibody to ensure mixing (if many conditions, consider making a master mix of antibodies)
    1. FITC-CD24:  2 µl per 100K cells (Isotype: FITC-IgG2A)
    2. PE-CD49f: 2 µl per 100K cells (Isotype: PE-IgG2A use 0.5 µl/100K)
    3. APC-EpCAM: 1 µl per 100K cells (Isotype: APC-IgG1)
  2. Incubate tubes with antibodies in the dark at 4C for 20 min or at RT for 10-15 min
  3. Add 3 ml of PBS + 2% CS to each tube to wash; pellet cells.  Repeat if there is time but usually one large wash is sufficient.
  4. Keep cells on ice until analysis with the Calibur