Isolation of peripheral blood mononucleated cells (PBMC) from blood
Materials
Experimental Procedure
1. Dilute blood sample with PBS in a conical tube (ratio must be at least 1:1)
2. Underlay the diluted sample with a volume of Ficoll® that is equal to the original sample.
3. Centrifuge for 20 minutes (2000 rpm).
4. Harvest PBMC located at the interface of the PBS and Ficoll® layers into a fresh tube.
5. Fill the tube with PBS.
6. Centrifuge cell suspension 4-5 minutes (1000-2000 rpm) at 4°C, discard supernatant.
7. Resuspend the cell pellet in flow Cytometry staining buffer and perform a cell count and viability analysis.
8. Centrifuge cells as in Step 6 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
- PBS
- Ficoll® Paque
- Flow cytometry staining buffer
- 15 or 50 mL conical centrifuge tubes
Experimental Procedure
1. Dilute blood sample with PBS in a conical tube (ratio must be at least 1:1)
2. Underlay the diluted sample with a volume of Ficoll® that is equal to the original sample.
3. Centrifuge for 20 minutes (2000 rpm).
4. Harvest PBMC located at the interface of the PBS and Ficoll® layers into a fresh tube.
5. Fill the tube with PBS.
6. Centrifuge cell suspension 4-5 minutes (1000-2000 rpm) at 4°C, discard supernatant.
7. Resuspend the cell pellet in flow Cytometry staining buffer and perform a cell count and viability analysis.
8. Centrifuge cells as in Step 6 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