I am trying to stain nuclei of Hela cells using Hoechst 33342. I have tried several working concentrations ranging from 0.2 ug/mL to 1 ug/mL for minimum 2 mins up to 10 mins. I obtain a weird staining at the cell membrane along with the nuclear signal (images attached).
Exptl. conditions-
I seed Hela cells on to poly-D-lysine coated coverslips. I tried adding Hoechst to live cells in culture media as well as adding Hoechst to fixed cells in PBS (+/- BSA) for the above mentioned conc. and time. I then wash the coverslips with PBS followed by mounting on to slide using Dako fluorescent mounting medium.
My aim is to study the localization of fluorescent lipids, that's why I can not use detergent like Triton.
Hi Bhumika,
Some cell lines can transport hoechst out of the cytoplasm. I suspect you are seeing the dyes that have been pumped out of the cell and are aggregating around the membrane.
Amit K Dash Hi Dr Amit, thats a good suggestion. I think DAPI staining of mycoplasma does not give such uniform ring-like pattern, it's usually found everywhere, inside and outside the cells. In my case, there is no change in growth rate, no abrupt change in media color, so I have ruled out presence of mycoplasma.
Mark Tingey Besides Hela cells, I have observed this pattern with U2OS cells too. I think it is some hidden technical problem, which I am not able to catch.
Bhumika do your live cell staining with Hoechst also gives this pattern?
Amit K Dash No, live cells do not give such pattern. I know of only Paraformaldehyde as a fixative for lipid microscopy. Methanol/acetone are not recommended. Could you please suggest any other method?
Bhumika if it is not in live cells then I think you have to modify the procedure mainly fixation but I donot know any other method of fixation for lipids other than formaldehyde. You have to search.
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