I have done DAPI staining in untreated and drug-treated EA carcinoma cells using 96-well plate. The procedure included only pbs washes, fixation with paraformaldehyde and incubation of each wells with DAPI (50ul of 1:1000 dilution). Haven't done any permeabilization process, but, still got stained images. Is it proper to do so?
Hello Ameena Stellixir
Permeabilization of cells is not absolutely required for staining, as DAPI will eventually enter the cells. However, this step of permeabilization will permit staining within minutes of incubation.
After fixing the cells with 4% (v/v) PFA final concentration, I incubate for 20 min at room temperature (20–25 °C). Then I wash the cells twice with 1X PBS. I perform the permeabilization step by dispensing 50 μl of 0.3% (v/v) Triton X-100 in each well, and incubating the plate at room temperature for 8-10 mins.
I would suggest that you include the permeabilization step in your protocol.
Best.
Hello Ameena
I have attached the paper for your reference.
Article Cell cycle staging of individual cells by fluorescence microscopy
Please see the subheading “Procedure”.
Good Luck!
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