DNA Content using Hoechst 33342 in Unfixed Cells
Purpose
To analyze DNA cell cycle in viable cells. Excellent for
simultaneous detection of DNA and GFP expression while still determining cell
viability.
Reagents
Cells to be
studied. If expressing green fluorescent protein (GFP), note that the same cell
type without GFP is needed as control.
Hoechst 33342
stock solution (1mg/ml) (see recipe)
12 X 75 mm culture
tubes
Vortex mixer
Water bath at 37oC
Method
1. Count cells.
2. Place approximately 106 cells
into a 12 x 15 mm test tube and spin them down by centrifugation for 5 min at
300 x g.
3. Remove supernatant by aspiration or rapid
decanting and add 500 μl of the medium that was used for growing the cells
to be studied pre-warmed to 37oC to the cell pellet. Mix gently. Add
5 μl of Hoechst 33342 stock solution and mix again. Incubate at 37oC
for 45 min.
The optimal Hoechst dye concentration and
staining time for different cell types vary as dye up-take depends on cellular
metabolic rates; thus, both have to be determined empirically. In general, dye
concentrations between 1μg/ml and 10 μg/ml and incubation times
between 20 min and 90 min will produce DNA histograms with acceptable
coefficients of variation. Because Hoechst DNA staining is performed on unfixed
cells, it is possible to use other non-vital DNA dyes, e.g., PI,
7-aminoactinomycin D (7-AAD), for concurrent dead cell discrimination.
Preparation of Hoechst 33342 stock
solution:
Dissolve 1 mg of Hoechst 33342 powder
(Molecular Probes, Eugene, OR) in 1 ml of distilled water. Store at 2-8oC
protected from light for up to 1 month.
Reference
Schmid I. and
Sakamoto KM. Analysis of DNA content and green fluorescent protein expression. In:
Current Protocols in Cytometry, Vol 1, Robinson JP, Darzynkiewicz Z,
Dean P, Orfao A, Rabinovitch P, Stewart C, Tanke H, Wheeless L, eds., John
Wiley & Sons, 2001, pp. 7.16.1-7.16.10.