We want to figure out optimal Hoechst concentrations for imaginal disc work. What has been reported in the literature is all over the place both in terms of stock concentration and final concentration. Also whether the stock solution should be in water, PBS or DMSO!!! So here are a few unorganized notes copied from other places, that should be used to figure things out.
In Drosophila protocols book (CSHL 2000), chapter 1, pg 10. They used it at 0.5ug/mL for mitotic cells in larval brains for fixed tissues. For live tissues they used 0.2ug/mL. Note (see below under Hoechst) that DAPI is more toxic than Hoechst.
DAPI concentrations anywhere from 0.1µg/mL to 10µg/mL final concentration https://www.thermofisher.com/ca/en/home/references/protocols/cell-and-tissue-analysis/protocols/dapi-imaging-protocol.html -Also, it's good to note, as Matthew Larkins said, that DAPI/Hoescht works at a range of concentrations. If you're interested in chromatin compaction, for instance, high concentrations will only result in an entirely blue kind of blown-out looking nucleus, while low concentrations will reveal differences between chromocenters, interchromatin space, heterochromatin and euchromatin. -I use 1.5ug/ml but added to mounting media so I don't incubate or wash. I just add the mounting media and cover slip.
Notes about hoechst concentration for various protocols
Very useful primers on using Hoechst dyes here and here. Some takeaways.
- Dissolve Hoechst in water or DMSO to make stock solution, but not PBS.
- Hoechst 33342 has better cell permeability than Hoechst 33258.
- Hoechst is less toxic to cells than DAPI, so better for FACS (?). Will not matter for fixed cells.
- Final concentrations recommended for fixed animal cells in the 0.2 - 2ug/mL range.
- Until further notice, I recommend a final concentration of 1ug/mL for work with our imaginal discs (fixed).
Hoechst (stock concentrations either 1mg/mL or 10mg/mL). typically dilute Hoechst stock 1:2000 or so. 0.2 - 2 µg/ml final(?) - the range of 1-10 µg/mL https://www.thermofisher.com/ca/en/home/references/protocols/cell-and-tissue-analysis/protocols/hoechst-33342-imaging-protocol.html
From the FACS protocol from the Edgar lab Final concentration is 0.5ug/mL) made from a 500ug/mL stock solution (dissolved in DMSO.. check for antibody staining). The reasoning for DMSO is that when it is made in a PBS based solution it would precipitate out of solution.
From the Drosophila protocols book (CSHL 2000) Chapter 1, pg 10. They used Hoechst 33258 at 0.5ug/mL. However this was to look at mitotically dividing cells at much higher resolution than we typically do.
From Drosophila: A practical approach 2nd ed. Chapter 6 (Embryos), they used Hoechst 33258 at a final concentration of 1ug/mL.
From this paper They used 1ug/ml in PBS for wing discs cultured in vivo
this paper incubated in 1ug/mL (diluted in PBS) for 5 minutes.
this paper DNA was stained for 5 min with 10 μg/ml Hoechst 33258 in PBS with 0.1% Triton X-100.
this paper Hoechst 33342 and rhodamine phalloidin, diluted 1:500 (Life Technology). I assume Life technology has a stock solution. check concentration.
This paper said they incubated at 1mg/mL for 30S (way higher than everyone else).