I would like to stain the cells with DAPI. I am using 24 well plate for cell culture. Is it possible to stain and observe under fluorescence microscope using the well plate directly? Could anyone help me to provide the detail protocol for it?
If the monolayer is still confluent, remove all the supernatent and let the cells a"bit" drying on air. Then fix it with ice-cold aceton-methanol (50:50) from -20°C. Add the nesessary amount of the fixans (and a bit more) to the monolayer and store the plate with it for 10 -20 min at -20°C. After that discharge the fixans, let the monolayer drying and add your DAPI solution. Normally it works perfect. We use it sometimes for DNA counterstaining. It is normally good to leave the plate at 4°C over night. After that you can observe the fluorescence because it is not "cloudy" in the solution anymore.
Have fun
Sven
Admitting not to have done such by myself but citing other sources:
see:
https://www.rndsystems.com/resources/protocols/protocol-preparation-and-fluorescent-icc-staining-cells-coverslips;
scroll down to: 'Protocol for the Fluorescent ICC Staining of Cultured Cells on Coverslips' find Point #8.
or, cf. : https://www.researchgate.net/profile/Mark_DeCoster/publication/8413953_Quantification_of_sPLA2-induced_early_and_late_apoptosis_changes_in_neuronal_cell_cultures_using_combined_TUNEL_and_DAPI_staining/links/09e41504a35af1dfe0000000.pdf , find 4. Detailed Procedure, go to: 4.3. Fixation and staining of cell cultures: "For DAPI-staining....
or: cf.: https://www.thermofisher.com/at/en/home/references/protocols/cell-and-tissue-analysis/protocols/dapi-hca-protocol.html
Or: cf.: http://www.igsb.org/uploads/pdf/ProtocolDAPIStaining.pdf
Or: cf: http://www.biotek.com/resources/articles/automated-tissue-culture-cell-fixation-staining.html
and there may be more out there in the wild ....best wishes and good luck...
Article Quantification of sPLA2-induced early and late apoptosis cha...
If the monolayer is still confluent, remove all the supernatent and let the cells a"bit" drying on air. Then fix it with ice-cold aceton-methanol (50:50) from -20°C. Add the nesessary amount of the fixans (and a bit more) to the monolayer and store the plate with it for 10 -20 min at -20°C. After that discharge the fixans, let the monolayer drying and add your DAPI solution. Normally it works perfect. We use it sometimes for DNA counterstaining. It is normally good to leave the plate at 4°C over night. After that you can observe the fluorescence because it is not "cloudy" in the solution anymore.
Have fun
Sven
Hi,
It depends on the microscope and the plates you are using. First you need an inverted microscope. Second, most plastic 24 well plates have a bottom that is too thick for the microscope. But there are microscopy plates with a thin glass or plastic layer which can be used directly for microscopy, I recommend you to use these.
Svens protocol above is one I also used before and it works nicely. But you can actually stain your living cells as well, just add the DAPI in the right concentration to the medium, incubate for 20 minutes, remove the medium and replace it with fresh one (preferably medium without the red indicator, it gives you less background) and you can do live cell imaging on your samples. If you want to preserve the cells afterwards you can use the fixation described above or a PFA (4% formaldehyde in PBS) fixation for 20 minutes at room temperature. Wash 2x with PBS and it is done.
Good luck!
Tilo
Thanks a lot Tilo but the DAPI solution contains methanol, normally. This is also a kind of fixation but with medium. Therefore I think it is more easy to fix it immediately by aceton-methanol at -20°C. When you compare the IFAT afterwards done with you protocoll and ours, you can see the difference. In our hands all plates from Corning (6, 12, 24, 48 and 96 well) are working well. There is no need to include an additional cover clip or a similar matrix for the cells. Also the fixation with PFA is a bit triggy. In our hand the IF was alsways a bit "swampy" and unclear, not really only located in the nucleus of the cells.
Best wishes and thanks for comments
Sven
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