1. Dissect tumor and mince with a clean (not necessarily sterile) razor blade
2. Scoop minced tumor into a 50 cc conical tube
3. Add 5 mL of  1x collagenase in DMEM/high glucose 10% CS
4. Incubate 3 hr rotating at 37°C
5. Manually dissolve cells by titurating with a glass pastuer pipet 3x
6. Spin down cells at 1000 rpm for 5 min
7. Resuspend cells in 1 mL .25% Trypsin EDTA
8. Incubate 10 min rotating at 37°C
9. Add 9 mL of DMEM/high glucose 10% CS
10. Run cell suspension over a 40 mm cell strainer (BD), rinse flow through with (ample) 1x PBS, so that the cells are suspended in no less than 30 mL of fluid
11. Spin down cells at 1000 rpm for 5 min
12. Resuspend cells in 10 mL 1x PBS, 3% CS and count by hemacytometer (with Trypan Blue if desired)
13. Spin down cells at 1000 rpm for 5 min
14. Resuspend cells at 1×10⁶ cells per mL in 1x PBS, 3% CS
15. Add 20 ml/mL of H2K-MHC1-PE and incubate 30 min at 4°C
16. Wash cells by adding 5x volume 1x PBS, 3% CS, spin down at 1000 rpm for 5 min
17. Resuspend cells at no more than 10×10⁶ cells/mL (less is better, they tend to clump)
18. Add 1:10,000 dilution of 1 mg/mL PI
19. Sort living (PI neg) H2K-MHC1-PE neg cells
20. Prepare sorted cells for flow analysis as described above.  You may want to add PI to the flow analysis as the ESA antibody will non-specifically bind to any permeablized cell; however using PI changes the way the CD24-PE fluorescence looks on a flow dot plot and therefore does not lend itself to publication worthy data.  Best to do both if you are worried about cell death from the previous FACs
    
    

Alternate Method for Analysis of Human Xenograft Tumors (no sorting)

1. Prepare cells as described in steps 1-14 above
2. Incubate cells with 10 ml/mL EpCAM-unconjugated antibody for 1 hr at 4°C
3. Wash cells by adding 5x volume of 1x PBS, 3% CS, resuspend at 1×10⁶ cells/mL
4. Add 10 ml/mL rat-anti-mouse-PerCP, incubate for 30 min at 4°C
5. Wash cells by adding 5x volume of 1x PBS, 3% CS, resuspend at 1×10⁶ cells/mL
6. Add CD24-PE, CD44-APC, and H2K-MHC1-FITC as described above
7. Incubate 30 min at RT
8. Flow the cells and gate out H2K-MHC1-FITC positive cells
   

Antibodies:

ESA-FITC, AbD Serotec, MCA1870F

CD24-PE, BD, 555428

CD44-APC, BD, 559942

H2K-MHC1-FITC, BD, 553569 (or PE for sorting, 553570)

Rat-anti-mouse-PerCP, BD, 340272

EpCAM, AbCam, ab8667-250