Hi all,
I just attempted a DAPI staining and got absolutely no results from the microscope. The obviously answer would be to increase the DAPI concentration, however I was wondering whether DAPI also required permeabilisation?
My current protocol is:
- Remove media
- Wash twice with PBS (for 5 minutes each)
- Fixation with 4% PFA (for 20 minutes)
- Wash three times with PBS (for 5 minutes each)
- 300nM DAPI (for 10 minutes)
- Wash three times with PBS (for 5 minutes each)
Should I be permeabilising the cells as well? I've been trying to research it online and some protocols say yes, but others say no.
In my experience, DAPI is only semi-permeant to live cells. Some cell lines label well, others partially, others not at all. So I always recommend at least a light permeabilization to insure good labeling. Hoechst 33342 (0.4 ug/mL for 5 minutes) is a much better option, as it is always permeant and labels the same way as DAPI (minor groove intercalator with dsDNA) with the same spectrum.
Also, your concentration is only half what I would recommend (0.2 ug/mL, = 600 nM) for a label time of 5 minutes in PBS.
In my experience, DAPI works best with permeabilised cells. Hoechst is weakly permeable and you would get some staining with that.
Dear Rosemary,
DAPI labelling does not require neither permeabilisation nor fixation. One can label nuclei of live cells. Therefore if you have problems with DAPI labelling you should look into other factors such as viability (health) of your cells without damaged nuclear membranes and extensive leakage; also check if the DAPI you are using is still good and able to label nuclei of healthy cells.
best wishes,
Refik
Technical Report Research Methods and Technical Considerations: Answers to ov...
I agree with Refik and I would make sure that the DAPI is very well dissolved in the solution you are going to use. Make sure that the solution which is used to make DAPI-stock is ok with the working solution DAPI. It is easy to see that: just pipette the amount of DAPI you need in the working solution and check if the drop dissolves or is as a small cloud. Good luck ;-)
I also agree With Refik. DAPI staining Works With both living and fixated cells. You can shorten Your protocol dramatically by adding the DAPI to the living cells (directly in the medium or with your working-solution), then fix directly and wash. Also, your washing steps of 5 minutes can be reduced: add the PBS, shake the dish carefully and remove right away.
Good luck! Cheers, Tilo
In my experience, DAPI is only semi-permeant to live cells. Some cell lines label well, others partially, others not at all. So I always recommend at least a light permeabilization to insure good labeling. Hoechst 33342 (0.4 ug/mL for 5 minutes) is a much better option, as it is always permeant and labels the same way as DAPI (minor groove intercalator with dsDNA) with the same spectrum.
Also, your concentration is only half what I would recommend (0.2 ug/mL, = 600 nM) for a label time of 5 minutes in PBS.
I agree with Jason. In my hands DAPI requires permeabilization and, in fact, I've used it as a dead cell indicator for some cells. I use DAPI at around 1 ug/mL. Hoechst 33342 is highly permeable (
For immunofluorescence microscopy, I use 300nM. If your method of detection is flow cytometry, try 3uM DAPI. I would recommend 15 minutes of DAPI treatment. I have tried HEK293T, THP1, J774, and Cos7 with just fixation and it has worked fine for me. I have never permeabilized my cells for DAPI staining.
Dear Rosemary,
I do not know the objective of your experiment. But I would highly recommend a product from Thermofischer Scientific (Invitrogen) called NucBlue Live ReadyProbes reagent which works beautifully on live cells (without fixation or permeabilization) for confocal microscopy. This reagent is a formulation of Hoechst 33422 and has the same spectral properties as H-33422.
Good Luck!
https://www.thermofisher.com/order/catalog/product/R37605
- Badges
- Science method
- Similar topics
- Biological Science
- Histology
- Staining