Cell Preparation from Lymphoid Tissue
Materials
Experimental Procedure
1. Harvest tissue (spleen, lymph nodes, thymus) into a tissue culture dish and press with the plunger of a 3 ml syringe.
2. Collect cells in 10 mL of flow cytometry staining buffer and pass cell suspension through a cell strainer to eliminate clumps. Collect cell suspension in a conical tube.
3. Centrifuge cell suspension 4-5 minutes (1000-2000 rpm) at 4°C, discard supernatant.
4. Resuspend the cell pellet and perform a cell count and viability analysis.
5. Centrifuge cells (as in Step 3) 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
- 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 (spleen, lymph nodes, thymus) into a tissue culture dish and press with the plunger of a 3 ml syringe.
2. Collect cells in 10 mL of flow cytometry staining buffer and pass cell suspension through a cell strainer to eliminate clumps. Collect cell suspension in a conical tube.
3. Centrifuge cell suspension 4-5 minutes (1000-2000 rpm) at 4°C, discard supernatant.
4. Resuspend the cell pellet and perform a cell count and viability analysis.
5. Centrifuge cells (as in Step 3) 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