ListServ Archive: Cell Staining

Cell Staining

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Does any one know a cell viability assay that works best for dicty? Has anyone tried commercially available kits like Promega’s CellTiter- Glo® Luminescent Cell Viability Assay? Also, let me know the assays for comparing cell motility and cell proliferation in dicty. Thank you very much.
-Sandhya Baviskar, Idaho State University , Feb 20, 2006

  • For cell viability we use Trypan Blue Exclusion just like with higher eukaryotic cells.
    -Daphne Blumberg, U. Maryland

  • I use a timelapse video setup on a microscope; you can watch ~ 50 cells for 10- 15 minutes and do this a few times; if the cells are putting out pseudopods and are motile I figure they're probably alive... We use the same system for motility (use NIH image to track the cells) and for proliferation we count.
    -Richard Gomer, Rice University

  • Molecular Probes Inc. now I think a part of invitrogen, has a kit which uses fluorochromes Ethidium Bromide derivative EthD-1 and calcein for cell viability assay. I had modified it to my requirements and it worked well. You may go through their catalog for other assay kits too. best wishes
    -Bhavesh Vats, Pharmaceutical Education and Research Development Centre

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We would like to be able to visibly stain amoebae adhered to plastic dishes to aid in the quantitation and imaging of clonal foci such as those obtained by transformation. Maintaining sterility or viability is not critical for our purposes. I would be grateful for any suggestions. Thanks in advance.
-Dale Hereld, University of Texas-Houston Medical School, TX, 6 Jul 2004

  • Thanks to all who replied to my question about staining foci of cells adhered to plastic. Generally, staining with crystal violet or coomassie blue concurrent with or following fixation was suggested. The suggestions I received are summarized below. Following Ann Hitt and Richard Gomer's suggestions, we directly stained with coomassie blue stain for protein gels (~0.03% coomassie R-250, 3% acetic acid, 50% methanol) and got staining adequate for imaging. Thanks again.
    -Dale Hereld, University of Texas-Houston Medical School, TX, 20 Jul 2004

    EXERPTS FROM REPLIES:

  • Jonathan Franca-Koh wrote: "Have you considered fixing the cells and then staining with crystal violet? Here is an example protocol I found on the web [http://www.whitelabs.org/Lab%20Protocols/Mammalian%20Cell%20culture%20and%20manipulations/Crystal%20Violet%20Staining.htm]. Washing with DB should be fine and cells could also be fixed with paraformaldehyde if preferred."

  • Thomas Winckler wrote: "we used the follwing protocol to stain clones. However, the stainings were rather light and hard to photograph.
    1. remove medium
    2. fix in cold solution 30 min at 4 degrees (83.5 ml water, 10.0 ml PBS, 5.7 ml formaldehyde, 0.8 ml glutaraldehyde)
    3. remove fixing solution
    4. add 20 ml Accustain (Sigma) undiluted
    5. stain over night at room temperature
    6. rinse with PBS until background is clear"

  • Paul Fisher wrote: "My virology colleagues tell me they use crystal violet (ca 0.2%) after formaldehyde (5%) fixation (to count viral plaques in a cell monolayer)."

  • Anne L. Hitt wrote: "In the past, I used Coomassie Blue stain (protein gel stain) and the protein gel destain. I don't know how quantitative it is, but it seemed to work with developing cells.... destaining should be as quick as a rinse or two with destain, then into water, or just let the plate air dry."

  • Richard Gomer wrote: "I just pour out the liquid and add coomassie, then rinse with water"

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Does anyone know a procedure for DNA staining in living Dicty cells? I have tried the fluorescent dyes Hoechst 33342 and 33258 but in my hands both do not stain the nucleus. Is there any special trick to use Hoechst 33342 and 33258 in Dicty?
-Ralph GrŠf, UniversitŠt Muenchen, Germany, 28 Sep 1999

  • Hoechst 33342 is exported out of Dicty cells, probably through an ABC-transporter mechanism (Irene Tatischeff has reported the presence of non-cellular, Hoechst staining bodies in cultures of Dicty). I observed a subpopulation of prestalk cells that retained Hoechst, but never did anything with that.
    I have heard anecdotal stories of using DAPI as a live cell DNA stain; perhaps the newer DIPI, with its affinity for AT, may be a better stain. Has anyone tried Molecular Probes' SYTO-16 on dicty?
    -Ricky Sucgang, Baylor College of Medicine, Houston, TX, 28 Sep 1999

  • I have tried a range of Syto dyes from the Molecular Probes sample set and generally none stained nuclei of live cells in my hands. I had it work once, and could not repeat it, so I suspect there is a trick that I haven't re-created. Someone reported one of them working at a past Dicty meeting, but I could not find specific information when I went back to check.
    -Dave Knecht, U. Connecticut, 28 Sep 1999

  • I have also tried the Syto dyes and had little to no luck. I also found that the critters didn't seem to like it at all, so even if there was some semblance of staining, there was no crawling. Alas, you can't study motility on a cell that doesn't crawl.
    -Deborah Wessels, University of Iowa, IA, 28 Sep 1999

  • We have used DAPI to stain living cells. You have to use electroporation to drive it across the membrane but once in the cell it will stain the nucleus and mitochondria very nicely. The cells seem to be fine for at least a couple of hours but after that the mitochondria appear to swell and the cells start looking funky. Good Luck,
    -Mary Kimble, 28 Sep 1999

  • I have done it with DAPI, so I am surprised that Hoechst 33258 does not work. The concentration is critical and should not be higher than about 0.1 µg/ml. Sometimes a fresh solution works well, sometimes an older one. We prepare the working solution fresh from a 100x stock (always in dist. water). The problem then is how do you visualize the stain? The dyes fluoresce under UV light, which kills the cells, unless you hit them with a very low dose and use video-enhancement. Good luck!
    -U.-P. Roos, University of Zurich, Switzerland, 29 Sep 1999

  • Try DAPI at 1 µ/ml for 5 to 10 minutes, rinse with medium twice before observation.
    -Yoshio Fukui, Northwestern University Medical School, Chicago, IL, 29 Sep 1999

  • I know well how difficult it is to stain nuclei into living Dictyostelium cells both with Hoechst 33342 and with DAPI ! The more alive the cells are and the more difficult is the task !!... If you really don't wish to fix the cells before staining, you can first work with Hoechst 33342 as a vital stain in (HL5) growth medium. Then, you can improve the nuclear staining by a "some hours" stay at 4°C: the cells are still alive but probably somewhat less healthy (!), so nuclear staining is improved. However, it remains still unhomogeneous from cell to cell. You can also immediately wash the cells out of the growth medium containing Hoechst 33342 and, then, fix the cells with 70% MeOH in buffer. Following the fixation step, nuclei become fully accessible to the stain which accumulated inside the living cells, without probably being able to reach the nuclei. It gives a nice homogeneous staining of the nuclei, following the vital staining of the cells....but, of course, these cells are dead "for ever"! Therefore, it might be a way to measure the cellular capacity for internalizing Hoechst 33342, but if your goal is to further work with living cells, you have to find a better trick!!! Good luck and thanks for sending me the final result of your investigation.
    -IrŹne Tatischeff, Universite Pierre et Marie Curie (Paris VI), Paris, France, 30 Sep 1999

  • Here is my protocol for vital staining of AX-4 cells with Hoechst33342. I have not seen any problem so far with Hoechst staining. Only trick that I could notice is to wash cells before staining, because HL-5 contains bunch of fluolescent materials which make annoying background staining. If you still have problems, please let me know. Good luck!
    Reagent: Hoechst 33342, M.W. 615.99 (Molecular Probes; cat. #H-1399)
    Stock Solution: 3mM Hoechst 33342 in H2O
    Staining Conditions:
    1. Wash cells once with K-PO4 20mM pH 6.5
    2. Resuspend the cells in K-PO4 buffer containing 20 mM Hoechst and incubate for 20-30 minutes at room temp. with occasional aggitation.
    3. Observe under fluorescent microscopy (UV excitation)
    -Shin-Ichi Matsuyama, Baylor College of Medicine, Houston, TX, 28 Sep 1999

  • I've just tested the method of Dr Matsuyama yesterday using 20 µM Hoechst 33342 and it works. However, at least with the lamp and filters I use, the fluorescence in viable cells is quite weak but visible whereas it is strong in dead cells. Thanks again for the stimulating discussion
    -Ralph GrŠf, UniversitŠt Muenchen, Germany, 1 Oct 1999

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Is Molecular probe's TOTO dye is able to stain LIVE gram negative bacteria or is there any other RED (or any color other than green) fluorescent dyes capable of doing the job??? I would also like to know if Cy3 dye is able to stain live bacteria! And is electroporation the usual method to induce the uptake of the fluorescent dyes into living bacteria, or one could just mix and incubate the dye and bacteria together for a period of time? By the way, I have asked at the Molecular Probes and they told me the only current dye capable of staining gram neg bacteria has only green emission!
-Ronnie Lee, ABI Institute of Cell Biology, Muenchen, Germany, 29 Sep 1999

  • Why not express the red-shifted form of GFP?
    -Rick Firtel, UCSD, CA, 29 Sep 1999

  • A very effective red dye (from Molecular Probes) is hexyl-rhodamine. Dynamically stains all membranes, cells remain alive!! Works beautifully with dicty in our hands. Also stained uneaten E.coli. Gives a vivid confocal image clearly separable from FITC. This dye is preferentially lipid soluble so you want leave a slight endogenous excess to keep loading the cell- there is no background fluorescence! original use was preferential staining of mitochondria with very low short-time doses of h-r. Loading of organelle and up to whole cell ("background") staining is dose and time dependent.
    -Jared Rifkin, Queens College, NY, 29 Sep 1999

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