FlowSortingFibroblastswithGFPandP.I.

• Protocol

1. Wash cells with PBS and trypsinize to a single cell suspension.

2. Count an aliquot on a hemocytometer. Meanwhile centrifuge the cells (e.g. 1000 RPM, 5 min.) to pellet them.

3. Resuspend the cells to a concentration of 2 x 10⁶ cells/sample with Flow Sort solution.

4. Aliquot into flow sort tubes (maximum 3 mL per tube)

5. Aliquot 1.5 mL of media into falcon tubes for collection.

6. Place cells and collection tubes on ice and transport to flow facility. Also bring transfer pipets and extra tubes.

7. Run untransfected cells first and set up voltage for GFP (FL1) and PI for viability (FL3).

8. Sort GFP(+), PI (-) cells. The maximum sort rate is about 5,000 total cells/second.

9. Sort for a maximum of one hour.

10. Spin down cells and plate (10⁴/cm² or 10⁶ cells/P100).

Materials

• 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)

• PBS

• 1x Trypsin/EDTA

• Media

• Flow sort tubes: Falcon #352054

• Sterile transfer pipets