Recently, I used the Hoechst 33258 to stain the nuclei of HepG2 cells. I did not fix the cells, because I want to combine the Hoechst staining with Calcein AM staining later.
I thought the Hoechst 33258 could stain all the cells, including live and dead cells. But I found some cells are not stained with Hoechst 33258, or they show very low fluorescence signal. The staining results are attached with bright field image and fluorescence image.
My staining protocols:
(1) Prepare Hoechst 33258 stocking solution----1mg/ml in H2O.
(2) Add 20μl Hoechst 33258 stocking solution into 2mL cell culture medium as working solution.
(3) Add 200ml working solution into cell culture plate (96-well plate).
(4) Incubator for 30min
(5) Wash wells with fresh cell culture medium and see the result under microscope
Thank you very much!
Hoechst 33258 is reported to be less cell permeable than Hoechst 33342 (look at product description in https://www.thermofisher.com/order/catalog/product/H3569), so probably the latter would be more appropriate for your assay since live cell staining is important for it.
The live cells may be excluding the dye due to the presence in the plasma membranes of efflux pumps, such as P-glycoprotein. Try adding an efflux pump inhibitor, such as cyclosporin A or reserpine.
Thank you very much for giving me suggestions! But I could not find good method, so far. The difference between Hoechst 33342 and 33258 is little. The result has no difference.
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