I would like to check cell cancer viability before and after treatment with specific drugs.
I use the protocol from life technologies (see link below). You'll need MTT powder, SDS and a plate reader that can read at wavelength 570nm. You can seed the cells at densities ranging from 1000-10,000 cells in 96 well plate (this will vary depending on which cell line you're using and their rate of proliferation). You can add the drug upon seeding the cells. Allow cells to proliferate for 1-3 days, then add MTT for 4 hours followed by SDS overnight.
http://tools.lifetechnologies.com/content/sfs/manuals/mp13154.pdf
The answer to the MTT part of your question has been answered well. Regarding the question of how to simply assess viability, you can stain with propidium iodide (1ug/mL) for at least 10 minutes at the end of your drug treatments. If the cells have died and the membranes have ruptured, then PI will stain as it is a vital stain. You can counterstain with Hoechst 33342 to stain nuclei of all cells for normalization (5-10ug/mL for 10-30 minutes usuallly works and can be done at the same time as PI staining). No special buffers required.
This doesn't account for any cells that have died via apoptosis but have not ruptured, but it is quick and dirty. This method though assumes you have a fluorescent scope you can use to check staining. Using this method, I've just taken images of several fields and counted the nuclei using ImageJ.
The other way is to use the same stains but do FACS analysis.
I frequently use TMRE in combination with a platereader (facs or microscopic analysis works fine also) - stains mitochondria of viable cells only. Used it for evaluation of nanoparticle toxicity on 80-90% confluent celllayers and since then im a fan because of it being cheap (~100€ for 25mg powder @ sigma) and easy to use.
Live Dead staining is a nice and easy way of checking cell viability: (http://www.lifetechnologies.com/order/catalog/product/L3224)
MTT is another easy assay that will give you information on cell metabolism (and cytotoxicty):
https://www.lifetechnologies.com/order/catalog/product/M6494?ICID=search-product
Life technololgies do kits for these, protocols can be found on their product pages (see links)
I give you a easy MTT protocol which allows to avoid the use of MTT kits :
* Prepare MTT solution at 5 mg/ml in PBS 1X (MTT Ref : Thiazolyl blue tetrazolium bromide Sigma M2128-5G). Sonicate solution for complete solubilisation. Store the solution at 4°C (for 1 week max) or at -20 °C (for 1 month max). Calculate the volume of MTT solution to prepare in function the test to do (100 µl solution for 1 culture well of 1 ml).
* Add 100 µl of MTT solution per culture well (well of 1 ml in 24 well culture plate). Prepare at least 6 culture wells with MTT but without your drug test for a negative MTT test.
* Incubate 4 h at 37 °C
* Wash 1 or twice the culture well with 500 µl PBS 1X
* Add 1 ml acidified isopropanol per culture well (Acidified isopropanol solution : for 100 ml : 0.3 ml Hydrochloric acid at 37% + Isopropanol) and 200 µl SDS 3% solution (3 g SDS in 100 ml ultrapure water).
* Incubate 30 min at room temperature
* Homogenize cell culture well by pipetting
* Take 3 samples of 200 µl of the previous cell culture well suspension and deposit each of them in a 96 well plate.
* Read the absorbance (DO) at 560 nm (time acquisition : 1 s)
* With the results obtained, calculate by using Excel software % viability of each sample as DO sample / DO negative sample (as described before) x 100.
% toxicity = 100 % - % viability.
Reference : Mosmann T (1983) J. Immunol Meth., 65, 55-63
Sincerly yours
Hello Jain Navodita
I do not dilute cell culture well suspension before taking the reading. Il work with 24 well cell culture plates and I never had absorbance saturation at the reading
Regards
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