I am working with a herbal extract and currently attempting to determine the appropriate concentration to use for further work, provided it does not cause cell-death- using cellular viability for determining the same.
Unfortunately, my extract is reacting with MTT, leading to the formation of formazon (as evidenced by the colour-change even in the absence of cells). It would be great if any of you could recommend an alternative protocol to check cell-viability (apart from trypan blue assay).
Thanks!
Hello Shalini Sanyal
There are several cell viability assays. They are grouped as follows.
Tetrazolium Reduction Assays
A variety of tetrazolium compounds have been used to detect viable cells. The most commonly used compounds include: MTT, MTS, XTT, and WST-1. These compounds fall into two basic categories:
1) MTT which is positively charged and readily penetrates viable eukaryotic cells.
2) MTS, XTT, and WST-1 which are negatively charged and do not readily penetrate cells. The latter class (MTS, XTT, WST-1) are typically used with an intermediate electron acceptor that can transfer electrons from the cytoplasm or plasma membrane to facilitate the reduction of the tetrazolium into the colored formazan product.
You may not be able to use the above assays as your herbal extract could react with tetrazolium salts.
As an alternative you can use:
1. Resazurin reduction assay:
Resazurin is a cell permeable redox indicator that can be used to monitor viable cell number with protocols similar to those utilizing the tetrazolium compounds. Resazurin can be dissolved in physiological buffers (resulting in a deep blue colored solution) and added directly to cells in culture in a homogeneous format. Viable cells with active metabolism can reduce resazurin into the resorufin product which is pink and fluorescent. For instance, the Alamar Blue assay.
2. ATP Assay:
The measurement of ATP using firefly luciferase is the most commonly applied method for estimating the number of viable cells. ATP has been widely accepted as a valid marker of viable cells. When cells lose membrane integrity, they lose the ability to synthesize ATP and endogenous ATPases rapidly deplete any remaining ATP from the cytoplasm. The ATP detection reagent contains detergent to lyse the cells, ATPase inhibitors to stabilize the ATP that is released from the lysed cells, luciferin as a substrate, and the stable form of luciferase to catalyze the reaction that generates photons of light.
The ATP assay has the advantage that you do not have to rely on an incubation step with a population of viable cells to convert a substrate (such a tetrazolium or resazurin) into a colored compound. This also eliminates a plate handling step because you do not have to return cells to the incubator to generate signal. For instance, you can use commercially available CellTiter-Glo® Luminescent Cell Viability Assay.
3. The sulforhodamine B (SRB) assay:
Sulforhodamine B, an anionic aminoxanthene dye, can form an electrostatic complex with the basic amino acid residues of proteins under moderately acidic conditions, which provides a sensitive linear response with cell number and cellular protein measured at cellular densities ranging from 1 to 200% of confluence. The SRB assay possesses a non-destructive and indefinitely stable colorimetric end point, and the color development is rapid and stable and is readily measured at absorbance between 560 and 580nm.
Good Luck.
For most purposes in our laboratory staining by nuclear dyes may be used. Dyes, which we use are Hoechst 33258 5 mkg/ml or Acridine orange 1 mkg/ml with 0.04 mkg/ml.
Cells are stained on cover slips or in 96-weel plates and photographed in phase contrast and fluorescence DAPI filtre (for both of dyes) and from orange to green (for Ethidium bromide). The percentage of cells is calculated. If you have flow cytometer, you may use it, but concentration of dyes need be corrected.
Good luck!
Would something like this work for you?
Article CellTiter-Glo™ luminescent cell viability assay: A sensitive...
Hello Shalini Sanyal,
A resazurin-based dye reduction assay would prove to be very easy, less time-consuming, and economical. For cell viability assay, prepare a stock solution of Resazurin (10mg/ml) in sterile PBS and prepare a working solution by diluting the dye 100 times (eg: Add 50ul dye to 4.950 ml of PBS). Simply add 20-30ul of this dye to each well (experiment and controls) and incubate at 37 degrees for 2 h. In case your cells are live they will reduce the dye from dark blue color to bright pink color. Take absorbance values at 610-620 nm and quantify your cells for viability/death using the standard formula used in MTT. This works really well and has also been reported in several papers to test the cytotoxicity of nanoparticles and compounds.
Hope this helps you!
Thank you Sergej Vladimirovich Nikolajev , Andreas Benesic , Jatin Chadha and Malcolm Nobre for your responses. I am currently attempting it with trypan-blue- as I already have the reagent at hand and receiving a good result, but it is time-consuming. Hoping to attempt some of the methods suggested here once I procure the necessary reagents to conduct the experiment with the same!
Dear Jatin Chadha , Thank you for the suggestion, and especially for the brief protocol ! :-)
- Badges
- Science topic
- Similar topics
- Microbiology
- Assays