Flow Sorting Fibroblasts with GFP and P.I.
M. Fero — 3/02
- Wash cells with PBS and trypsinize to a single cell suspension.
- Count an aliquot on a hemocytometer. Meanwhile centrifuge the cells (e.g. 1000 RPM, 5 min.) to pellet them.
- Resuspend the cells to a concentration of 2 x 106 cells/sample with Flow Sort solution.
- Aliquot into flow sort tubes (maximum 3 mL per tube)
- Aliquot 1.5 mL of media into falcon tubes for collection.
- Place cells and collection tubes on ice and transport to flow facility. Also bring transfer pipets and extra tubes.
- Run untransfected cells first and set up voltage for GFP (FL1) and PI for viability (FL3).
- Sort GFP(+), PI (-) cells. The maximum sort rate is about 5,000 total cells/second.
- Sort for a maximum of one hour.
- Spin down cells and plate (104/cm2 or 106 cells/P100).
- GFP transfected cells
- Untransfected cells as negative control
- Flow Sort solution: 25% media, 75% PBS with 2µg/mL propidium iodide, 10 µg/mL DNAse (or 10 U/mL)
- 1x Trypsin/EDTA
- Flow sort tubes: Falcon #352054
- Sterile transfer pipets