We seeded 2,000 Dermal Papilla cells in 96 well-plate and treated the cells
for 24, 48, and 72 hrs in order to check the proliferative effects of our compounds.
Then we stained them with 0.05% crystal violet at the different time points and we found a purple plaque as shown in the image.
I would like to ask that
1. What is the plaque??
2. Where does the plaque come from?
You need to indicate how your cultures were processed; were they fixed, if so with what fixative? It's difficult to suggest anything w/o knowing what was done to the cultures.
Especially what was done with the cell culture....and, BTW, it would be of benefit for any commenter to have the right margin of the image (= the margin of the seeding well= i.e., where that strange 'morphology' appears) at a higher magnification, HIGHLY-resolved image.... TYIA.
(it 'seems' that there has been formed kind of 'dermal' sheet=epidermal layers, but this also could be a certain roughness / wall inhomogeneity of the well )...
NB: Q in addition: is there any specific background or necessity why you use Crystal violet for staining? Is it in vivo staining (means: seeded cells expected to be / stay alive for further experiment -as I'd guess to be their 'fate' - or is your staining done AFTER initial histological processing (like fixation with?, dehydration? etc....) again: TYIA...
Are you looking directly at the cells or are the samples processed and mounted with a coverslip?
Wolfgang H. Muss
Sheldon R Gordon
Sorry for the late reply, the ambiguous questions, and the poor English writing.
For the image, I took it directly from the 96-well plate. In my in vitro experiment, I have to check my compound whether they are able to induce cell proliferation at 24, 48, and 72 hours. So, I seeded Dermal Papilla at 2000 cells/well in "new" 96-well plates and treated them with my compounds. Then, After subjecting the cells to the treatment for 24, 48, and 72 hours. I stained the cells with 0.05% crystal violet in 4% formaldehyde for 20 minutes. After washing them with water three times, I found that there were some plaques as shown on the right side of the picture.
I would like to know
-Where do the plaques come from? Substances produced by cells or problems of dry??
-If I aim at checking the cell proliferation, would it be better to change to MTT assay to avoid getting false positive results from the plaques?
Thank you
After looking at your reply and the image shown I would suggest that you fix your tissues, rinse them well, then stain with cresyl violet, and differentiate the stain with distilled water. If you can, completely air dry your cells and mount and coverslip them. Newer cresyl violet from several manufacturers washes out when passed through both water and ethanol to dehydrate samples. The older Kodak cresyl violet never had this problem but I don't think they make it any longer. In some ways your image reminds me of slides that were mounted in Permount that were not completely dehydrated.
Since after all I did NOT get any notice (as usual) by RG-System that I was mentioned by Narawat Nuamnaichati and came back to this particular thread only "accidentally" right now I'd like to inform you, that I shall get back to this issue as soon as possible (have to leave within next 5 minutes).
problem solving not quite easy, I guess !
You should be use a soultion of crystal violet in ammonium oxalat1:10 staining.
A solution:
Crystal violet:2gr
Absolute ethanol 100cc
B solution:
Ammonium oxalat:1gr in 100cc DW
C: 1cc A additive to 9cc DW
1cc C+4cc B
Staining cell cultures after fixed with 7o%ethanol for
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