Hello Good people
When I stained my adherent non-transfected cells with Hoechst 33342 staining it showed blue fluorescence but dull staining happened with GFP transfected cell
I used 2ug per molar
30 min incubation at RT
300ul per well in 12 wells plate
So, what's your suggestion for better procedure to be able to see cell segmentation more clearly!
What's the benefits from PBS washing as recommended by some protocols at the beginning or the end!
No I just took both GFP and Dapi channels together automatically from the fluorescence microscope but I checked the DAPI channel alone it was dull not like the non transfected cells
Hello Nicolas,
1- I read that fluorescent dyes like (PI.EB,DAPi)are impermeable through the cell membrane of viable cells except for Hoechst dyes.That 's why I haven't done permeabilization just removing media then adding stain dye for 30 min at RT then removing the stain and mounting the cultured mono layer adherent cells on slides and visualized using a fluorescent microscope for direct two colors blue and green analysis .
2- I prepared for example 10 ml of 2ug/ml then I froze them down as a liquotes in -20c and I used the same a liquotes the only difference that I used for non transfected cells 300 ul and for transfected 200 ul in each 12 wells plate.
Doaa Yamani
Are you doing live or fixed cell imaging?
Always make a fresh batch of Hoechst solution, and always mix Hoechst with cell media. (avoid using PBS).
Hoechst is cell permeable, in contrast to DAPI. The uptake of Hoechst is cell dependant, and you may need to optimize the concentration for the cell line being used.
From the image attached, you may need to lower the power or exposure time of your GFP channel, or increasing the time between each channel excitation, it may also be a focusing issue.
Washing steps are critical: wash x3 with PBS (1-2 min for each wash)
Thanks Hossam for your feedback
Im using fixed cells with Hoechst diluted with PBS!
Im doing washing step from 5-10 mins!
Doaa Yamani
For fixed cells you can use PBS, but you need to permeabilize your cells with Triton X100 at 0.1%, followed by PBS wash (x3), then blocking using BSA (5%), and PBS wash (x3). Now you can add your Hoechst dye solution in PBS for (10-30min depending on cell line).
Hope that helps.
Recently Im using your suggested reagents and concentrations but does it make difference if im using for example BSA 3 %
Thanks :)
Doaa Yamani
The duration of the blocking step is more important and I wouldn't worry about concentration used, from my personal experience 1-5% BSA is fine.
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