PombeNet
Goin' fission....

Forsburg Lab Protocols: Staining cells with DAPI or Hoechst



DAPI staining fixed cells: we have used three methods. These can be used on live cells (less efficient), or on fixed cells. If not treating for immunofluorescence, we find that ethanol fixation (as done for FACS) works quite well, as does heat fixation performed by putting a small aliquot of culture on a slide and exposing it briefly to a hot plate.

DAPI Method I:


A 10x DAPI mounting stock may be prepared and aliquoted. This is 10µg/ml DAPI, 10mg/ml p-phenylenediamine . (The DAPI stock is 1mg/ml in DMSO. ) Take a 20µl aliquot and add 180µl 100% glycerol . This gives a 1X working stock (1µg/ml DAPI, 1mg/ml p-phenylenediamine which acts as anti-fade, 90% glycerol) which should be kept at -20°C in the dark.

DAPI Method II:


A working stock of 0.5µg/ml of DAPI in PBS may be used to wash the cells for 30-60". They are then washed free in PBS. A mounting medium is made from a stock of 10mg/ml p-phenylenediamine in 1M Tris and diluted 1:9 in 100% glycerol.

DAPI Method III:


Another mounting stock is made as follows:
50mg n-propyl gallate
50mg p-phenylenediamine (PPD)
Dissolve in 5mls PBS. Add glycerol to 50mls. Aliquot and store in freezer in the dark. If it turns dark, discard. Add 1µl DAPI stock per 1.5mls (final concentration is .66µg/ml), store frozen and dark.
Live cell staining
You can use DAPI, but it doesn't get into live cells quite as well. Hoechst 33442 has been recommended as a more effective live cell nucleic acid stain. When we get the conditions and concentrations, we will add them here.

Hoechst


We have had good results using the live-cell Hoechst 33342 protocol described in Hiraoka et al. Chromosoma (2000) 109 p104.
They describe washing defined media out of cells then staining for 15 min at room temperature with 0.5 ug/mL Hoechst in water followed by resuspension in defined media. We find this protocol to be quite tolerant. Concentrations of Hoechst upwards of 1 ug/mL produce nice staining, although it is expected any Hoechst-DNA staining will be toxic within a cell cycle or two.
Staining in water produces superior results to adding stock Hoechst to cells in media, but the latter will also work.

If cells are not being maintained, they can be imaged in the water-Hoechst staining solution.
Dead cells will take up Hoechst with an order better efficiency, to the point that they will compromise an image. Care should be taken to ensure a culture to be stained is maximally healthy and mid-log phase. We have seen some indication that cells in stationary phase resist staining.

Cells grown in rich (YES) media show very inferior staining.