EpCAM CD24 CD49f Cell Line Flow (Click to Download)
- Collect cells by trypsinization to generate a single cell suspension
- If needed, DNase I treat and/or filter cells through a 40 µm or 20 µm mesh to ensure removal of clumps
- Resuspend cells at 1 x 106 cells/ml in PBS + 2% calf serum
- 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
- 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)
- FITC-CD24: 2 µl per 100K cells (Isotype: FITC-IgG2A)
- PE-CD49f: 2 µl per 100K cells (Isotype: PE-IgG2A use 0.5 µl/100K)
- APC-EpCAM: 1 µl per 100K cells (Isotype: APC-IgG1)
- Incubate tubes with antibodies in the dark at 4C for 20 min or at RT for 10-15 min
- 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.
- Keep cells on ice until analysis with the Calibur