How to determine the optimum concentration of dye

The best concentration for Calcein-AM and PI depends on the cell type, so it is necessary to determine the concentration when staining each cell. The best concentration can be determined using the following protocol.

Optimum concentration for PI (red, PI is a possible carcinogen)

·         Stain the desired cells with 0.1-10 μmol/L of PI. Too high a concentration of PI will stain not only nuclear but also cytosol, staining concentration should be adjusted to the appropriate range.

·         Fix the cells prior to staining using one of the methods below if necessary:

o   Treat the cells for 10min with 0.1% saponin or 0. 1-0.5% digitonin.

o   Treat the cells for 30min with 70% ethanol.

Optimum concentration for Calcein-AM (green)

Using the fixed cells to stain with 0.1-10 μmol/L of Calcein-AM solution. Determine the concentration range that will not stain all of the fixed cells. Next, using the living cells, determine if the concentration is enough to stain the cells. If sufficient staining has not been obtained, increase the concentration of Calcein-AM.

Q: What should the power-type reagent be dissolved in?

A: Please dissolve the reagent in DMSO for viable cell staining reagents. Since DMSO easily absorbs moisture, please use fresh DMSO. After the preparation of the DMSO solution, aliquot in an appropriate volume and store at -20C. Working solutions prepare using PBS (-) are not stable enough to store. Discard the remaining working solution after use.

Q: Among all the staining reagents used for living cells, which one remains the longest inside the cells?

A: CFSE remains relatively the longest inside the cells. It has been reported in a paper that the fluorescent dye was retained within cells for up to 8 weeks. Also, the fluorescents of Calcein-AM and BCECF-AM have been observed in cells for up to 3 days. (PMID: 2212694, PMID: 8300411).

Q: Which staining reagents used for living cells have the lowest cytotoxicity?

A: Calcein-AM and BCECF-AM seem to have the lowest cytotoxicity.

BCECF-AM: This was originally used to measure pH inside the cell, and is also used as a dye to stain living cells.

Calcein-AM: this has the least effect on cell function.

Other things about Calcein-AM: Calcein is stable both inside and outside of the cell. However, Calcein-AM is not stable. If there is excess Calcein-AM on the outside of the cell, the excess may break down and fluoresce, which would be a source of error. It is necessary to remove by washing. Fluorescence intensity is affected by a fluctuation in pH, so it is necessary to set the pH conditions.

Why is it not possible to use Calcein-AM assays that contain serum?

A: Calcein-AM is broken down on the outside of the cell by serum in the media. In order to make it possible to do an essay with a small number of cells, remove anything that may cause the background fluorescence.

CFSE: after entering into a cell, it combines with the amino base of protein in the cell membrane on the cytoplasm side. As a result, it leaks out of the cell comparatively less than other dyes.

FDA: The oldest known dye. It leaks out of the cell relatively quickly.

Q: What are the differences between the nucleus staining reagents AO, Hoechst 33258, and Hoechst 33342 other than fluorescent wavelength?

A: The differences are listed below:

AO: it is possible to distinguish between single-stranded DNA and double-stranded DNA using the difference in fluorescence wavelength when intercalating with a double-stranded DNA and when combining with the phosphoric acid of a single-stranded DNA. AO passes through the membrane of living cells. (Maximum wavelength at 526nm)

Hoechst 33258 and Hoechst 33342: Binds specifically with adenine- thymine base pairs of DNA. They pass through the cell membrane and stain the DNA of living cells. Hoechst 33342 has a higher membrane permeability. Better staining is possible when cells are fixed.

Other nucleus stains:

PI: It does not have base specificity. It binds to all DNA and RNA, but the fluorescence intensity is higher when intercalating and can be used widely among a variety of cells. Ethidium Bromide (EB) and PI molecules intercalate inside the DNA double helix.

DAPI: This will bind with the minor groove of a double chain, and has a high affinity for adenine-thymine base pairs.