Measurement of Green Fluorescent Protein Expression and DNA Content in Unfixed Cells
Reagents
Cells to be studied 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
Waterbath 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 ml 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 ml 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 1mg/ml and 10 mg/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.
Cells to be studied 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
Waterbath 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 ml 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 ml 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 1mg/ml and 10 mg/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.
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