Fix cells with formaldehyde

1. Count cells (appendix 3a).

See unit 9.12 for retroviral transduction of red-shifted GFP into cells and see Kain (2000) for strategies for cell transfection.

2. Place ~106 cells into a 12 x 75-mm test tube and wash once with phosphate buffered saline (PBS) by centrifuging 5 min at 300 x g, 2° to 8°C.

Preferably, the PBS should be cold (4°C); however, this is not absolutely necessary as the cells will stop growing when exposed to UV light.

3. Remove supernatant by aspiration or rapid decanting and add 500 |l cold (~4°C) PBS to the cell pellet. Mix gently. Add 500 |l cold (~4°C) fixation solution and mix again. Incubate 1 hr at 2° to 8°C.

Different concentrations of formaldehyde fixative may be needed for optimal retention of red-shifted GFP in various cell types, and for obtaining acceptable coefficients of variations on DNA histograms as discussed below (see Commentary).

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