Mycins (7-Actino-, Chromo-, 7-Amino- )

SYTOX

DAPI

TOTO

Hoechst

TO-PRO

BOBO

YOYO-1

LDS-751

DRAQ5

Oli Green

PI

SYTO

7-actinomycin-D / 7-Aminoactinomycin D / Chromomycin A3

Chromomycin A3 works fine in fixed cells as a green fluorescent DNA dye.  On the other hand, 7-Actinomycin D, available from Sigma or Molecular Probes, is a good choice for red staining of the nucleus. It binds selectively to GC pair in DNA, and you'll get NO specific fluorescence in RNA-containing structures,  a lot easier than propidium iodide or any other Feulgen dyes. Normally use paraformaldehyde or methanol for fixation and stain cells at 10 uM of 7-AAD for 30 min.

Time required for staining is longer than for DAPI. Weaker fluorescence than DAPI.

7-Aminoactinomycin D, which can be excited by 568 nm line of Kr/Ar laser and emits red fluorescence (>610 nm).  This DNA dye just works fine in fixed cells without needing any permeabilisation or digestion procedures, just as DAPI in UV mode

BOBO

BOBO dye will also work for the 568 line, but it gets knocked out of the DNA rapidly during excitation. 

DAPI

         1. Equilibrate cells in PBS.

2. Dilute DAPI 1:300 to 1:1,000 with PBS.

3. Incubate cells for up to 5 minutes.

3. Wash 3x with PBS.

Hoechst

Hoechst 33258 is useful for looking at chromosomes in developing

sea urchin eggs.  As long as the dye concentration was kept very low I was

able to see the chromosomes and the fertilized eggs developed normally to at

least 16 cell stage. 

LDS-751

LDS-751 on live HEK 293 cells:

1.      Make 5 micro molar solution in normal medium from a 5 mM DMSO stock.

2.      Incubate at 37°C for 30 mins,

3.      Wash 4x with physiological saline.

The stain is membrane permeant and has an excitation/emission maximum of 543/712 (!) nm. despite its extreme stokes shift it's fully usable with filter setup equal to Texas red. I've had essentially no problems with RNA/cytoplasmic staining, probably because there is a significant red/blue shift in the ex/em  to 590/607 upon binding to RNA. It's also fairly cheap

Oli-Green

Works well with the 488 line

PI

Propidium iodide - used it to stain nuclear DNA and to quantify it. You do have to get rid of the RNA in the cytoplasm. PI is a DNA intercalater, so it will label all double stranded nucleic acids. We added DNase free RNase from Boehringer at 25 microgram per ml to the propidium aiodide solution. Use at 1.3000 concentration.

         1 If the sample is not already in 2X SSC, equilibrate briefly with 2X SSC.

2 Dilute propidium iodide (Component C) 1:300 in 2X SSC; e.g., 1 µL added to 300 µL of 2X SSC is usu-ally enough stain for one coverslip preparation. Incubate cells, covered with the dilute stain, for 1–5 minutes.

3 Rinse samples several times in 2X SSC. Drain excess buffer from coverslip and mount in a medium such as Cytoseal, SlowFade, SlowFade Light or ProLong.

RNase treatment is recommended when formaldehyde fixed samples are to be counterstained with SYTOX Green or propidium iodide. RNase treatment is not necessary in the methanol/glacial acetic acid, acetone or acetone/Triton X-100

protocols (see sections 4–6). Because DAPI is highly DNA-selective in our staining conditions, even when formaldehyde fixation is involved, RNase is not usually required.

1 Equilibrate samples briefly in 2X SSC.

2 Incubate in DNase-free RNase, 100 µg/mL diluted in 2X SSC, for 20 minutes at 37°C.

3 Rinse samples 3–4 times, for 1 minute each, in 2X SSC.

SYTO

In blood vessels I can report that SYTO-13 (1uM) gave the best results (i.e. very bright and doesn't bleach too quickly)

with Syto14 which very well penetrated live cells and binds to DNA and RNA. . However Syto14 was rather toxic to our cells and two hours after using the dye all cells were dead

only Syto 16was not toxic for more than 24h in our CHO cell line that our cells only survived for 18h (our purpose) with Syto16  on H540 leydig tumor cells and found that Syto16 was not toxic during at least 24h. However when I tried to do some time‑lapse imaging with our CLSM I found that the laser dye

combination is very critical for the cells. Low laser levels and a single layer image every 30 min was possible for 5 to 6 hours. Syto 16 could also be used with MA‑10, R2C, CHO and Cos cells.

Syto 23 seems to be the brightest of all the Syto stains.  I don't have much toxicity experience yet, but we did a trial on cultured brainstem slices.  The slices lived 48 hrs after 1 hour labelling in a 1/1000 dilution from the stock reagent

SYTOX

SYTOX (R) from Molecular Probes (www.probes.com) works like a very bright green Propidium Iodide.  That is to say:

     1. it can't get into "live" cells

     2. it stains the nuclei of permeablized cells

     3. I use fluorescein filters with the 488 line and it works great

  We have tried Sytox BLUE on formalin fixed cells and it does NOT work (Excessive background fluorescence!). Upon inquiry Molecular probes told us that Sytox Dyes in general don't work on aldehyde fixed cells or tissues. It worked nice though on unfixed preparations.

  1. If the sample is not already in 2X SSC, equilibrate briefly with 2X SSC.

   2. Dilute SYTOX Green (Component B) 1:300 in 2X SSC; e.g., 1 µL added to 300 µL of 2X SSC is usually enough stain for one coverslip preparation. Incubate cells, covered with the dilute stain, for 1–5 minutes.

   3.  Rinse samples several times in 2X SSC. Drain excess buffer from coverslip and mount in a medium such as SlowFade, SlowFade Light or ProLong reagents.

TOTO

TOTO-3 from Mol. Probes, which excites in the far red region, on a BioRad 1000, and I am getting very nice results. In some fixation regimes it also stains (very weakly) the cytoplasm, probably because it also binds RNA

TOTO- 3 works well for the a 633 or 647 laser line. Note that all of these work extremely well on formaldehyde fixed cells and tissues, as well as milder alcohol or acetone fixes.  Cell and tissues most be permeabilized as these are impermeant dyes.  It is also important to treat with DNAse free RNAse, as all of these will label RNA as well. 

TO-PRO

YO- and TO-PRO also stain RNA. Use RNAse to degrade cytoplasmic RNA before staining. TO-PRO-3 from molecular probes.  It is far red, seems to stain only the nucleus, and is very bright.

To-Pro-3 iodide works well on fixed preparations (sharper on methanol- than PFA-fixed preparations, and slightly beeter after RNAse treatment - probably because the DNA is more compact).

  We use a 1:200 dilution

  The disadvantage is that the dye bleaches rapidly, penetrates relatively slowly, is not particularly intense in thick specimens and of course stains only in the dark red.

YOYO-1

YOYO-1 works well for the 488 line

DRAQ5

DRAQ5 works well with living cells as a Far Red nuclear stain. It is available from Biostatus Limited. It acts in the same manner as other DNA binding/intercalators and should be handled accordingly. To use with living cells, add directly to cell buffer or medium at a concentration of 1-20mM at room temperature. DRAQ5 is exremely cell permeant. Cells and tissues do not need to be permeabilized. To use with fixed cells, add directly to buffer and incubate for 15 minutes, rinse and mount with an anti-fade agent. Double labeling cells with DRAQ5 and FITC does not require fluorescence compensation as with propidium iodide. It has a wide range of laser excitations: 568, 633, and 647. Our 660LP emission filter produces lovely images.