(b) Adjust pH to 7.4 with 1 N NaOH.
(c) Add DNase to a concentration of 25 μg/mL (Stock aliquots are 1 mg/mL) fresh each time.
2. MACS Buffer: PBS solution with 0.5% bovine serum albumin
(BSA) and 2 mM EDTA. Must be degassed.
2.2 Other Reagents
1. APC donkey anti-rat IgG.
2. Brilliant Violet 421 goat anti-rat IgG.
3. Anti-rat IgG Microbeads for magnetic labeling of cells.
4. Collagenase IV.
5. 5 mg/mL 4
0 ,6- Diamidino-2-phenylindole (DAPI).
6. Rat anti-mouse E-cadherin IgG, Clone ECCD-2 (M108,
Takara, Clontech).
7. DMEM/F12.
8. Enzyme-free, Cell Dissociation Buffer.
9. Foxp3/Transcription Factor Staining Buffer Set (eBioscience).
10. KPCY [11] or similar strain of genetically engineered or
implantable tumor model in which cancer cells are fluorescently tagged.
2.3 Equipment
1. 6 cm dishes.
2. 10 cm dishes.
3. 96-well plates.
4. FACS machine with 100 μm nozzle.
5. Cell strainers (70 μm).
6. Conical tube (15 mL and 50 mL).
7. Transfer pipettes.
8. Fine scissors.
9. Luer Lok syringes (3 mL).
10. Microfuge tubes.
11. Table top microcentrifuge.
12. Needles (18 gauge).
13. Polypropylene round-bottom tubes (5 mL).
14. Polystyrene round-bottom tubes with 35 μm cell strainer cap
(5 mL).
15. Benchtop Refrigerator Centrifuge.
16. Vortexer.
17. Water bath.
18. 1.5 mL microcentrifuge tubes.
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Robert J. Norgard and Ben Z. Stanger
(c) Add DNase to a concentration of 25 μg/mL (Stock aliquots are 1 mg/mL) fresh each time.
2. MACS Buffer: PBS solution with 0.5% bovine serum albumin
(BSA) and 2 mM EDTA. Must be degassed.
2.2 Other Reagents
1. APC donkey anti-rat IgG.
2. Brilliant Violet 421 goat anti-rat IgG.
3. Anti-rat IgG Microbeads for magnetic labeling of cells.
4. Collagenase IV.
5. 5 mg/mL 4
0 ,6- Diamidino-2-phenylindole (DAPI).
6. Rat anti-mouse E-cadherin IgG, Clone ECCD-2 (M108,
Takara, Clontech).
7. DMEM/F12.
8. Enzyme-free, Cell Dissociation Buffer.
9. Foxp3/Transcription Factor Staining Buffer Set (eBioscience).
10. KPCY [11] or similar strain of genetically engineered or
implantable tumor model in which cancer cells are fluorescently tagged.
2.3 Equipment
1. 6 cm dishes.
2. 10 cm dishes.
3. 96-well plates.
4. FACS machine with 100 μm nozzle.
5. Cell strainers (70 μm).
6. Conical tube (15 mL and 50 mL).
7. Transfer pipettes.
8. Fine scissors.
9. Luer Lok syringes (3 mL).
10. Microfuge tubes.
11. Table top microcentrifuge.
12. Needles (18 gauge).
13. Polypropylene round-bottom tubes (5 mL).
14. Polystyrene round-bottom tubes with 35 μm cell strainer cap
(5 mL).
15. Benchtop Refrigerator Centrifuge.
16. Vortexer.
17. Water bath.
18. 1.5 mL microcentrifuge tubes.
318
Robert J. Norgard and Ben Z. Stanger