I am planning to measure DNA concentration of fixed mammalian cells on 6 well plates. I am using Hoescht for staining. Do I need to use any cell permeabiling agent before I stain them? Should I remove the stain and wash it with PBS? Do I have to measure them within PBS or I can dry the plate and then measure it by plate reader?
Hi,
Hoechst is a cell permeable dye therefore you do not need a permeabilization step before staining. 5 min of incubation with the dye before reading the plate should be enough. However, Hoechst staining is suitable to measure high concentrations of DNA and not appropriate for measuring lower concentrations (0-2 ng/ml). PicoGreen and SYBR Green I are reported to be more than one thousand times more sensitive than ethidium bromide and Hoechst 33528 (attached).
What do you mean by measure DNA concentration? If you are planning to just stain the nucleus/DNA with Hoechst and then do IF, then you don't need to fix/permeabilize cells. Dilute Hoechst to the desired concentration in PBS or H2O. Remove media from the cells and rinse once with PBS. Add the diluted Hoechst and then incubate for 15-30 minutes protected from light. Remove the staining solution and wash the cells 2-3X with PBS. Add PBS and then proceed to IF. You could use the same procedure to measure Hoechst signal using a fluorescence enabled plate reader. Do not dry the plate.
If you are going to measure the DNA content of the attached cells, you can seed them in dark cell culture plate then stain them with Hoechst and read the plate fluorometrically by a fluorometric plate reader. Finally compare them with control group and report the results as fold change in compare with control. But its not applicable in 6-well plate. For this purpose you must use fluorescence microscopy that is semi-quantitative technique
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