I want to do cell viability assay using trypan blue dye for my plant extract, I need a full protocol from start of tissue culture, then treatment with cell and then how to use trypan blue dye? Do we need to collect floating cell and the trypsinise remaining cells and then counting?
Just wondering whether you are actually trying to do a cell count in a "plant extract". A plant extract is a product of "extraction" of various compounds present in the plant cells which will have proteins, lipids and N number of other compounds.
Dear Erum Dow, scientific value of work with "plant extract" will be doubtful. Thousands of unidentified compounds presented in these extracts may have thousands of molecular targets in in vitro cultured cells. Many of these substances may be toxic for cultured cells or affect metabolism and survival nonspecifically. Unlikely output of these studies will result in obtaining any useful and conclusive data. The most probably, you will need to isolate and identify any substances (or groups of substances) from "plant extract" to make your work and obtained results sensible.
Thanks Rohitash Jamwal, i already have article one, and your article 2 is really helpful, but still it is not clear to me how would it works, as it is mentioned in article 2 that add 0.1ml trypan blue in incubated vials, then after that what to do? should i take some microliter and do counting in hemocytometer? in this case how i will able to count live cells?
Dow, page number 3 of the article 2 explains it very nicely. Hope this helps.
In vitro cytotoxicity studies
Viability staining by trypan blue dye exclusion method.
The aqueous extract was studied for short term in vitro cytotoxicity using human bone marrow cells (k562 cell line) and lymphocyte cells as normal cells. The cells were seeded in 96-well plates. Four wells for each concentration were seeded and triplicate plates were used the cell line. Then, the cells were incubated at 37°C. After 24 h the medium was replaced by fresh medium containing different concentrations of the plants extract. After incubation 0.1 ml trypan blue was added and number of dead cells determined by using haemocytometer. The percent viability was calculated by using formula:
% viability = (live cell count/total cell count)*100
Cytotoxicity assay.
Cytotoxicity of sample on tumor cells was measured by microculture tetrazolium
(MTT) assay [33]. For the assays, 96-well microplates were seeded with 100 μl medium containing 5, 000 cells. After 24 h incubation and attachment, the cells were treated with 6 fourfold dilution of crude extracts. Exactly from the stock solution (40 mg/ml), each extract sample was applied in a series of 6 dilutions (final concentrations ranging from 15.6 to 500 μg/ml) with a final DMSO concentration of 0.1% and was tested in quadruplicate. After 48 h incubation, cell viability was
determined by adding (Sigma) tetrazolim salt as cytotoxicity indicator, so after 48h of incubation, 15μl of MTT (5mg/ml) in phosphate buffered saline (PBS) was added to each well and incubated at 370C for 4h. The medium with MTT was then flicked off and the formed formazan crystals were solubilized in 100μl of DMSO and then measured the absorbance at 570 nm using micro plate reader. Tetrazolium salts are cleaved to formazan dye by cellular enzymes (only in the viable cells). The %
cell inhibition was determined using following formula.
% cell Inhibition = 100- Abs (sample)/Abs (control) x100.
The Inhibitory concentration required for 50% cytotoxicity (IC50) value was analysed using sigmaplot software.
The most probably, next step will be never, because very complex and hardly reproducible effects will be obtained in experiments with plant extracts. My advice to you: work with pure, well identified substances. If you search for anti-cancer therapeutics, do this in in vivo experiments.
If you are doing primary screening of plant extracts for anti-cancer activity, you may check the toxicity level initially on a cell line such as HeLa. Microscopic observation should suffice.
Hello Erum,
I will start the same test as you described.
Can you advise me what range of extract concentrations you used?
Also did you count cells using Trypan blue from each well of the 96-well plate?
