Staining

Aliquot 200K of cell (200 µl), if possible to 10 FACS tubes. (note not all stains need to be done on all tumors or samples, 10 tubes would be the max)

(note can use down to 100K for tumor single stains, may want to use up to 500K  cells for tumor triple stains/double stains if gating on GFP+ cells)

1. 1. Unstained
   2. Isotypes 24/49f/EpCAM
   3. Isotypes 24/44
   4. PI
   5. CD24 PE
   6. CD49f PerCP-Cy5.5
   7. EpCAM APC
   8. CD44 APC
   9. Triple:  CD24/CD49f/EpCAM
   10. Double:  CD24/CD44
2. Add all antibodies/isotype controls and stain for 20’ at 4C.
3. Add 2 ml ice cold FB, pellet cells.
4. Resuspend in 2 ml FB, pellet cells,
5. Resuspend in 250 µl FB (or more if more cells). Ice.
6. Add 25 ml 1:1000 PI (1mg/ml orginal) to PI tubes.

Antbodies used (amounts are for 100K cells):

CD24-PE (mouse IgG2A) BD Cat# 555428 clone ML5:  USE 4 µl

CD49f-PerCP-Cy5.5 (Rat IgG2A) BioLegend clone GoH3:  USE 1 µl

EpCAM-APC (mouse IgG1) BD Cat# 347200 clone EBA-1:  USE 1 µl

CD44-APC (mouse IgG2B) BD Cat#559942 clone G44-26:  USE 4 µl

Mouse IgG2A-PE isotype control BD Cat# 559319:  USE 4µl

Rat IgG2A-PE-Cy5 isotype control BD Cat# 551066:  USE 1 µl

Mouse IgG1-APC isotype control BD Cat# 340442:  USE 1 µl

Mouse IgG2B-APC isotype control BD Cat# 555745:  USE 4 µl

Flow data collection

Run control sample to set voltages and compensation with CD24/49f and EpCAM.

Double check and adjust voltages with one tumor sample, set compensation of GFP.

Set GFP+ gate, collect 30,000 GFP+ events but collect all data (in acquisition and storage) for unstained and double/triple stained. Note:  for single stains collect only 10,000 GFP+ events or 10,000 cells if this is still too slow.

Tweak compensation for PI and collect PI sample to check for dead cells.

Remaining Cells

Plate at 5000/ml for tumorsphere formation in non-adherent 6-well plates

Inject 100K per gland in 50% matrigel:media for passage of the tumors into adult non-cleared non-humanized female mice.

Freeze back leftovers…plate for cultures…toss?