DAPI Staining

DAPI is used to stain DNA and in our group is normally used to determine cell cycle information.
  1. In most cases the cells will be spun down and the supernatant removed before adding DAPI.
  2. Resuspend the pellet in at least 300 µl of DAPI. In general you would want the cells at a concentration of 2 x 106/ ml of DAPI. The cells can be run on the flow cytometer immediately or kept on ice in the dark for later in the day.
  3. The cells can also be frozen for analysis at a later date.
Fresh cells in suspension:

DNA Content and Cell Cycle Analysis
Analysis is performed as previously described (Rabinovitch, P.S., Reid, B.J., Haggitt, R.C., Norwood, T.H., and Rubin, C.E. Progression to cancer in Barrett's esophagus is associated with genomic instability. Lab. Invest. 60:65 71,1989). In brief, cells in suspension are spun and resuspended in a solution of 10µg/ml 4,6-diamidino-2-phenylindole (DAPI) and 0.1% nonidet P-40 detergent in a Tris buffered saline. The suspension is triturated with a 26 gauge needle and analyzed on the cytometer, with ultraviolet or violet excitation and DAPI emission collected at >450nm. DNA content and cell cycle are analyzed as previously described (Rabinovitch, P.S. DNA content histogram and cell cycle analysis. Meth. Cell Biol. 41:263-296,1994) using the software program MultiCycle (Phoenix Flow Systems, San Diego, CA).

Tissues:

DNA Content and Cell Cycle Analysis
Analysis is performed as previously described (Rabinovitch, P.S., Reid, B.J., Haggitt, R.C., Norwood, T.H., and Rubin, C.E. Progression to cancer in Barrett's esophagus is associated with genomic instability. Lab. Invest. 60:65 71,1989). In brief, tissue is minced with scalpels in a solution of 10µg/ml 4,6-diamidino-2-phenylindole (DAPI) and 0.1% nonidet P-40 detergent in a Tris buffered saline. The supernatant is triturated with a 26 gauge needle, filtered through 40µm steel mesh, and analyzed as above.

Cells from Paraffin:

DNA Content and Cell Cycle Analysis
50mm sections cut from paraffin-embedded tissue are processed by a variation of the technique originally reported by Hedley et. al. (Hedley DW, Friedlander ML, Taylor IW, Rugg CA, Musgrove EA. Method for analysis of cellular DNA content of paraffin-embedded pathological material using flow cytometry. J. Histochem. Cytochem. 31:1333-1335, 1983). In brief, sections are dewaxed in xylene, gradually rehydrated in a step series of ethanol solutions, and digested in water with 1% pepsin pH 1.5 37°C for 40 minutes. The supernatant is triturated with a 26 gauge needle and resuspended in an isotonic pH 7.4 buffered solution with 0.1% nonidet P-40 detergent, and 10µg/ml diamidino-2-phenylindole (DAPI), and filtered through 40mm steel mesh. The analysis is performed on a cytometer using UV or violet excitation. 50,000 cells are analyzed, if available, and in all cases acceptable histograms contained at least 10,000 cells and a coefficient of variation below 6.0%. DNA content and cell cycle are analyzed as previously described (Rabinovitch, P.S. DNA content histogram and cell cycle analysis. Meth. Cell Biol. 41:263-296,1994) using the software program MultiCycle (Phoenix Flow Systems, San Diego, CA).

1X DAPI (Working Solution)

To 500 ml dd H2O add:
8.5g NaCl (final conc. = 146 mM)
1.2 g Tris Base (final conc. = 10 mM)
Adjust pH to 7.4 with HCl.

Add:
4 ml of 500 mM CaCl2 solution (final conc. = 2 mM)
44 ml of 500 mM MgCl2 solution (final conc. = 22 mM)
50 mg (0.05g) BSA
1 ml nonident P-40 ** (Sigma I8896, Igepal CA-630) detergent (final conc. = 0.1%)
10 mg DAPI (4,6-diamidino-2-phenylindole*) powder
(final conc. = 10 µg/ml)
100 ml DMSO (final conc. = 10%)

Add dd H20 to final volume of 1 L.
Store in dark or foil wrapped bottle at 2-6°C.
*Accurate Chem. Co. #18860
** Igepal CA-630 is chemically indistinguishable from Nonidet P-40, which is apparently no longer commercially available. Tergitol Type NP-40 is NOT the same as nonidet p-40 and should not be used.