CELL VIABILITY and how to measure it.
CELL VIABILITY and how to measure it.
Read this first.
Then watch video
Then Read the Pubmed article about the section on MTT assay and graphs in Results.
Cell Viability typically refers to health status pf the human cells in culture. Assays to measure viability, proliferation,and cytotoxicity are commonly used to monitor the response and health of cells in culture after treatment with various stimuli. Cell viability and cytotoxicity assays are used for drug screening and cytotoxicity tests of chemicals.
Assays for cell proliferation may monitor the number of cells over time, the number of cellular divisions, metabolic activity, or DNA synthesis.
Cell counting using viability dyes such as trypan blue or calcein-AM can provide both the rate of proliferation as well as the percentage of viable cells.
MTT (3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide) is a water soluble tetrazolium salt yielding a yellowish solution when prepared in media or water. Dissolved MTT is converted to an insoluble purple formazan by cleavage of the tetrazolium ring by dehydrogenase enzymes. This water insoluble formazan can be solubilized using isopropanol and the dissolved material gives a blue-purple color measured spectrophotometrically yielding absorbance as a function of concentration of converted dye.
The cleavage and conversion of the soluble yellow dye to the insoluble purple formazan has been used to develop an assay. Active mitochondrial dehydrogenases of living cells will cause this conversion. Dead cells do not cause this change.
Routinely, MTT stock solution (5 mg/ml) is added to each cell culture being assayed to equal one-tenth the original culture volume and incubated for 3 to 4 hr. Then the converted dye may be solubilized with DMSO (Dimethyl sulfoxide). Absorbance of converted dye is measured at a wavelength of 570 nm with background subtraction at 630–690 nm.
Trypan Blue, another method.
Trypan Blue is one of several stains recommended for forviable cell counting. This method is based on the principle that live (viable) cells do not take up certain dyes, whereas dead (non-viable) cells do. Staining facilitates the visualization of cell morphology. –
Now watch this
https://www.youtube.com/watch?v=KoYpGaNvwi8 for MTT assay
Summary: Human cells are in each well of the 96 well plate with media (nutrients-red colored ). Some wells have cells without a drug (control) and some wells have cells treated with a drug to test if they grew less (growth inhibititon) or treated with a mitogen to test for more growth. MTT is added, incubated 4h and blue crystals are dissolved to read the color instensities. More purpleish color, more growth, more OD reading at 570 nm. All 96 wells are read together in an spectrophotometer called a Plate reader.
Watch this for Trypan Blue counting
https://www.youtube.com/watch?v=1YZT_HAgbrc (cut and paste, link may not work)
Summary: Live human cells in culture are placed on a special slide with coverslip, TB is then added, immediately observed under a microscope. Blue cells are dead or non viable. Blue and unstained cells are manually counted.
Now READ the paper on Curcuminhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3733655/pdf/1475-2867-13-64.pdf
Read it in the sequence below
Read ABSTRACT, BACKGROUND, and ONLY 1 paragraph of RESULTS with FIG 1 ONLY.
Read section in DISCUSSION relating to growth inhibiition
Read the methods for MTT assay. (in this paper, it comes in last)
SUBMISSION 1: MTT assay and studies using MTT
Write authors and title of article
1. MTT assay description; what it is used for, what happens in the cell, how is it viability measured.See Roche web site for help. What are advantages and limitations of MTT assay.
2. Take paper 1 with focus on your cell line, and dietary agent, and MTT result. Copy the table or the graph, explain what happened in the study. What cancer did you study? Use terms dose dependence or concdependence , or time dependent study if you see it.
3. Any out of the ordinary result is always exciting, so that can be explained. Show the graphs, discuss results.