Cell Preparation from Non-Lymphoid Tissue
Materials
Experimental Procedure
1. Harvest tissue and mince into 2-4 mm pieces using scalpel blade.
2. Add appropriate amount of enzyme(s) diluted in PBS and incubate at the optimal temperature for the appropriate amount of time according to enzyme manufacturer instructions.
3. Disperse cells by gentle pipeting and filter through a cell strainer to eliminate clumps. Collect cell suspension in a conical tube.
4. Centrifuge cell suspension 4-5 minutes (1000-2000 rpm) at 4°C, discard supernatant.
5. Resuspend the cell pellet in PBS to remove excess enzyme solution
6. Centrifuge cells as in Step 4.
7. Repeat steps 5 and 6.
8. Resuspend the cell pellet in staining buffer and perform a cell count and viability analysis.
9. Centrifuge cells as in Step 4 and resuspend in the flow cytometry staining buffer so that the final cell concentration is 1x107 cells/mL.
References: http://www.scbt.com/protocols/protocol_08.pdf
http://media.ebioscience.com/data/pdf/best-protocols/cell-preparation-for-flow-cytometry.pdf
- Scalpel
blade
- PBS or other suitable physiologic buffer
- 60 mm x 15 mm tissue culture dish
- mL syringe
- Falcon® cell strainer
- Flow cytometry staining buffer
- 15 or 50 mL conical centrifuge tubes
Experimental Procedure
1. Harvest tissue and mince into 2-4 mm pieces using scalpel blade.
2. Add appropriate amount of enzyme(s) diluted in PBS and incubate at the optimal temperature for the appropriate amount of time according to enzyme manufacturer instructions.
3. Disperse cells by gentle pipeting and filter through a cell strainer to eliminate clumps. Collect cell suspension in a conical tube.
4. Centrifuge cell suspension 4-5 minutes (1000-2000 rpm) at 4°C, discard supernatant.
5. Resuspend the cell pellet in PBS to remove excess enzyme solution
6. Centrifuge cells as in Step 4.
7. Repeat steps 5 and 6.
8. Resuspend the cell pellet in staining buffer and perform a cell count and viability analysis.
9. Centrifuge cells as in Step 4 and resuspend in the flow cytometry staining buffer so that the final cell concentration is 1x107 cells/mL.
References: http://www.scbt.com/protocols/protocol_08.pdf
http://media.ebioscience.com/data/pdf/best-protocols/cell-preparation-for-flow-cytometry.pdf