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Ch 8.Immunohistoch / immunology
Ch 10.GC/MS, NMR and Proteomics
Cell Viability and Proliferation Assays / XTT Cell Viability Assay Protocol
Description
: XTT Cell Viability Assay Kit provides a simple method for determination of live cell number using standard microplate absorbance
readers. Determination of live cell number is often used to assess rate of cell proliferation and to screen cytotoxic agents. XTT
is a tetrazolium derivative. Similar to MTT, XTT measures cell viability based on the activity of mitochondria enzymes in live cells
that reduce XTT and are inactivated shortly after cell death. Unlike the water-insoluble formazan produced from MTT, XTT is readily
reduced to a highly water-soluble orange colored product (1,2), thus omitting the solubilization step required for the MTT assay.
The amount of water-soluble product generated from XTT is proportional to the number of living cells in the sample and can be quantified
by measuring absorbance at wavelength of 475 nm.
Kit Components (Biotium)
5 bottles (10mL each) of XTT Solution (sterile)
5 vials
(50_L each) of Activation Reagent PMS (sterile)
Storage and Handling
Upon receipt the kit should be stored at -20¡ãC and protected
from light. Stored properly, the kit components should remain stable for 6 months.
Experimental Protocol
1. Plate cells
into 96-well tissue culture plates. In general, cells should be seeded at densities between 5000 and 10,000 cells per well since they
will reach optimal population densities within 48 to 72 hours.
2. Carry out your experiment by adding chemicals or biological
agents. The final volume of tissue culture medium in each well should be 0.1mL, and the medium may contain up to 10% Fetal Bovine
Serum.
3. Thaw one bottle of XTT Solution and one vial of Activation Reagent. Mix XTT Solution and Activation Reagent for two
96-well plates. For one 96-well plate, mix 25¦ÌL Activation Reagent with 5mL XTT Solution to derive activated XTT solution and save
the rest of XTT Solution and Activation Reagent for later use.
Note: If sediment is present in the XTT Solution, heat the solution
to 37¡ãC and swirl gently until a clear solution is obtained.
4. Add 25¦ÌL or 50¦ÌL of the activated XTT solution to each well
and incubate the plate in an incubator for 2-24 hours (usually, 2-5 hours is sufficient).
Note: 50¦ÌL activated XTT solution generates
a sharper increase of signal but reaches signal saturation at a lower cell number than 25¦ÌL activated XTT solution. 25¦ÌL activated
XTT solution gives a broader dynamic range detection.
5. Shake the plate gently to evenly distribute the dye in the wells.
6.
Measure the absorbance of the samples with a spectrophotometer (ELISA reader) at a wavelength of 450-500 nm. In order to measure reference
absorbance (for non-specific readings), use a wavelength of 630-690 nm.
Note :
1. Defrost and prepare the reaction mixture immediately
prior to use.
2. Since the test is extremely sensitive, it is possible to use a low concentration of cells in the wells (approximately
5000 cells per well). Since there are cell types that show low metabolic activity, such as lymphocytes, kartinocytes and melanocytes,
it is recommended to increase the concentration of cells to 2.5x105 cells per well, in order to obtain development of formazan color
within a reasonable period of time.
3. Incubation time with the reaction mixture varies according to the type and concentration of
the cells. Therefore, it is advisable to perform an initial test by reading the absorbance at various time lapses, i.e. after 4, 6,
8, and 12 hours using the same plate.
4. Prior to reading the absorbance with a spectrophotometer, the plate should be gently shaken
in order to evenly distribute the dye in the wells.
5. If the volume of the media in each well is larger than 100¦ÌL, add a larger
amount of reaction mixture by the same increment (i.e. 100¦ÌL reaction mixture to 200¦ÌL growth media).
In Vitro Toxicology
Assay, XTT based (TOX2) - Bulletin
IN VITRO TOXICOLOGY ASSAY KIT XTT BASED. Product Number TOX-2. Storage Temperature ¨C0 ¡ãC. Product
Description. This kit provides a means for determining ...
www.sigmaaldrich.com/sigma/bulletin/tox2bul.pdf
XTT Cell Proliferation--FAQs--t
The
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screening, ...
http://www.promokine.de/index.php?id=134
Protocol at-a-glance ... The use of XTT greatly
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www.bioind.com/Htmls/article.aspx?C2004=12557&BSP=12410