It is called Cell Viability and Proliferation

simply using the Counting Cells Using Hemocytometer

Or there are several methods such as

please visit

http://www.sigmaaldrich.com/technical-documents/articles/biofiles/cell-viability-and-proliferation.html

MTT

Cell Viability Applications

MTT (3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide; thiazolyl blue) is a water soluble tetrazolium salt yielding a yellowish solution when prepared in media or salt solutions lacking phenol red. Dissolved MTT is converted to an insoluble purple formazan by cleavage of the tetrazolium ring by dehydrogenase enzymes.1 This water insoluble formazan can be solubilized using isopropanol or other solvents and the dissolved material is 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 system alternative to the conventional 3H-thymidine uptake and other assays for measurement of cell proliferation. Active mitochondrial dehydrogenases of living cells will cause this conversion. Dead cells do not cause this change. This has been applied in measurement of interleukin-2 activity in a multiwell assay.2 Modification has improved the sensitivity.3 Other uses such as measurement of cytotoxicity4 and cell number have also been developed.

In our testing we dissolve MTT, (Cat. No. M5655), 5 mg/ml in RPMI-1640 without phenol red. This medium is available as a powder (Cat. No. R8755) or liquid (Cat. No. R7509). The solution is filtered through a 0.2 μm filter and stored at 2–8 °C for frequent use or frozen for extended periods.

Routinely, MTT stock solution (5 mg/ml) is added to each culture being assayed to equal one-tenth the original culture volume and incubated for 3 to 4 hr. At the end of the incubation period the medium can be removed if working with attached cells and the converted dye may be solubilized with acidic isopropanol (0.04-0.1 N HCl in absolute isopropanol). When working with suspension cells the dye is added directly and dissolution is accomplished by trituration. Absorbance of converted dye is measured at a wavelength of 570 nm with background subtraction at 630–690 nm.

MTT may also be used to score hybridoma development or clonal development. Clones will convert the dye and become readily visible without magnification. It should be noted that MTT is a mutagen and the resultant cells may be affected. The concentration used may be reduced by dilution of the stock solution (5 mg/ml) to 0.1 mg/ml and adding a tenth volume to each well. Incubation should be monitored by observing for stained clones. Cells can be recovered by gently washing the cells and adding growth medium.

Technique Summary

MTT stock solution: 5 mg/mL

Typical use: Add 1/10th of culture volume

Solvent: 0.04-0.1 N HCl in isopropanol

Can also use DMSO:isopropanol (1:1)

Spectrophometric reading: 570 nm

Background wavelength: 630-690 nm

Trypan Blue

Trypan Blue is one of several stains recommended for use in dye exclusion procedures for viable 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.

NOTE: Trypan Blue has a greater affinity for serum proteins than for cellular protein. If the background is too dark, cells should be pelleted and resuspended in protein-free medium or salt solution prior to counting.

Protocol for Viable Cell Counting using Trypan Blue

Prepare a cell suspension in a balanced salt solution (e.g., Hanks' Balanced Salts [HBSS], Cat. No. H9269).

Transfer 0.5 ml of 0.4% Trypan Blue solution (w/v) to a test tube. Add 0.3 ml of HBSS and 0.2 ml of the cell suspension (dilution factor = 5) and mix thoroughly. Allow to stand for 5 to 15 minutes.

Note: If cells are exposed to Trypan Blue for extended periods of time, viable cells, as well as non-viable cells, may begin to take up dye.

With the cover-slip in place, use a Pasteur pipette or other suitable device to transfer a small amount of Trypan Blue-cell suspension mixture to both chambers of the hemacytometer. Carefully touch the edge of the cover-slip with the pipette tip and allow each chamber to fill by capillary action. Do not overfill or underfill the chambers.

Starting with chamber 1 of the hemacytometer, count all the cells in the 1 mm center square and four 1 mm corner squares (see Diagram I). Non-viable cells will stain blue. Keep a separate count of viable and non-viable cells.

Note: Count cells on top and left touching middle line of the perimeter of each square. Do not count cells touching the middle line at bottom and right sides (see Diagram II).

Repeat this procedure for chamber 2.

Note: If greater than 10% of the cells appear clustered, repeat entire procedure making sure the cells are dispersed by vigorous pipetting in the original cell suspension as well as the Trypan Blue-cell suspension mixture. If less than 200 or greater than 500 cells (i.e., 20–50 cells/square) are observed in the 10 squares, repeat the procedure adjusting to an appropriate dilution factor.

Withdraw a second sample and repeat count procedure to ensure accuracy.

Calculations

Cell Counts – Each square of the hemacytometer, with cover-slip in place, represents a total volume of 0.1 mm3 or 10-4 cm3. Since 1 cm3 is equivalent to approximately 1 ml, the subsequent cell concentration per ml (and the total number of cells) will be determined using the following calculations:

Cells Per mL = the average count per square × dilution factor × 104 (count 10 squares)

Example: If the average count per square is 45 cells × 5 × 104 = 2.25 × 106 cells/ml.

Total Cells = cells per ml × the original volume of fluid from which cell sample was removed.

Example: 2.25 × 106 (cells/ml) × 10 ml (original volume) = 2.25 × 107 total cells.

Cell Viability (%) = total viable cells (unstained) ÷ total cells (stained and unstained) × 100.

Example: If the average count per square of unstained (viable) cells is 37.5, the total viable cells = [37.5 × 5 × 104] viable cells/ml × 10 ml (original volume) = 1.875 × 107 viable cells. Cell viability (%) = 1.875 × 107 (viable cells) ÷ 2.25 × 107 (total cells) × 100 = 83% viability.

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I've had good success with Alamar Blue as a proliferation assay.

It's very easy; you add to your cell culture, incubate, and read. If you're working in a plate and have a platereader you can just do the assay as is. If you're working in flasks or such, just add, then take a media sample to assay.

Here's the info from Life Technology:

http://www.lifetechnologies.com/pl/en/home/brands/molecular-probes/key-molecular-probes-products/alamarblue-rapid-and-accurate-cell-health-indicator.html#what

I think you can try the cyquant cell proliferation assay kit. This uses DNA binding dyes to measure cell proliferation. Very very accurate. Can measure cell number as low as 100-1000 cells.

You can use the EdU incorporation (invitrogen kit) or the Ki67 staining (abcam antibody). I use both of them and they work very good.

MTT is more for cell viability. I think BrdU/EdU, Ki67 and counting the cells are accurate measures of proliferation. Performing one type of staining, followed by counting your cells under similar conditions, would really confirm your proliferation studies.

I am also fan of very old but gold method - count the cells:-)

if you dont want to do the aforementioned ones ; try using Cell counting kit 8 (CCK8). it is pretty quick to do and reliable as well, and there is flow cytometry as well...

Proliferation index (%) =(S +G2/M)/(G0/G1 + S + G2/M)X100%.

I would recommend click-it EdU kit, Ki-67 staining, or CFSE/Celltrace Violet labeling and measuring proliferation by dilution of dye.

Ki 67 staining reveals all the cells which are outside G0 phase of cell cycle, whilst BrdU incorporation is a marker of DNA synthesis. Both are methods are largely validated for cell peroliferation measurements. Ki 67 is specific of human cells and for animal cells may be used PCNA staining which works very well in both human and animal samples.

Cfse- poliferation assay with flow cytometer measurement

http://www.jove.com/video/2259/the-use-carboxyfluorescein-diacetate-succinimidyl-ester-cfse-to

Hi Venumadhav,

Tritiated Thymidine incorporation is also one of the method for calculation of proliferation of cells.