Dear Mahmoud

You can perform MTT Assay. It is an easy and fast method.

http://www.atcc.org/~/media/DA5285A1F52C414E864C966FD78C9A79.ashx

Other methods:

Comet assay

DNA Fragmentation assay

Tryphan blue exclusion test for cell viability

DAPI Staining

EtBr-Acridine orange staining

A best and fast method for cytotoxicity is BST (Brine Shrimp Test).

Thank for you Rajaram and Zahra for you help. I did BST before but i ask for another method (fast and accurate).

SRB (Sulphorhodamine B) assay would be a good option to determine cytotoxicity of your samples.

Thanks Alok, could you send a procedure of SRB to determine the more active fractions or compounds for my samples.

SRB assay is a rapid, sensitive and inexpensive method for measuring the cytotoxic potential of test substances, based on the cellular protein content of adhered suspension cultures in 96 well plate. This method is suitable for ordinary laboratory purposes and for large-scale applications like high throughput in vitro screening in anticancer drug development.

Principle of assay:

The anticancer activity is determined by the cytotoxic potential of the test material using human cancer cell line, which is allowed to grow on tissue culture plate in the presence of test material. The cell growth is measured on ELISA reader after staining with Sulphorhodamine B (SRB) dye which binds to basic amino acid residues in trichloroacetic acid (TCA) fixed cells.

• This assay is carried out as described by Skehan et al. (1990).

• The SRB assay provides a sensitive method for measuring drug cytotoxicity in culture. In a pilot study of NCI’s in vitro anticancer drug discovery project, the SRB assay is used to examine the differential sensitivities of 60 human cancer cell lines to more than 1,000 test compounds.

• The method appears to offer several advantages over the MTT and XTT assays. It is simpler, faster and more sensitive than the MTT assay, provides better linearity with cell number, permits the use of saturating dye concentrations, and is less sensitive to environmental fluctuations. It is independent of intermediatory metabolism and provides a fixed end point that does not require a time sensitive measurement of initial reaction velocity.

• SRB is a bright pink aminoxanthene dye with two sulfonic groups. SRB binds to protein basic amino acid residues in TCA-fixed cells to provide a sensitive index of cellular protein content. Color development in SRB assay is rapid, stable and visible. The SRB staining method is nondestructive in the sense that it is not necessary to digest samples.

Procedure of assay:

• The plates are taken out from the incubator after 48 hours of adding test samples.

• To stop the reaction, gently add 50 µl of chilled 50% TCA (trichloroaceticacid) to each well of the plate, making final concentration of 10%.

• Incubate the plates at 4C for one hour to fix the cells attached to bottom of the wells.

• Wash the plates 5-6 times with distilled water.

• Plates are air-dried.

• Add 100 µl of SRB dye(0.4% in 1% acetic acid) to each well of the plate and leave the plates at room temperature for 30 minutes

• Wash the plates with 1% acetic acid after 30 minutes.

• Plates are again air-dried.

• Add 100 µl of Tris buffer (10.5 M) to each well.

• Shake the plates gently for 10- 15 minutes on a mechanical shaker.

• Record the optical density with ELISA reader at 540 nm wavelength and maintain the data

Calculations:

The viability and growth in the presence of test material is calculated as:

OD Change = Mean of OD of Test sample – Mean of OD of Blank

% Growth in presence of the Control = 100 / OD change in presence of control

% Growth in the presence of Test sample =

(% Growth of in presence of control) × (OD change in presence of test sample)

% Inhibition by the Test sample =

% Growth inhibition= 100 – [OD (test sample) - OD (blank)/ OD (control) – OD (blank)] ×100

T / C value for 50% growth inhibition for each test material is calculated from its three concentrations

Reference:

Skehan P, Storeng R, Scudiero D, Monks A, McMohan J, Vistica D, Warren JT. New colorimetric cytotoxic assay for anticancer-drug screening. J Natl Cancer Inst. 1990; 82: 1107–1112.

Regards

Thanks Alok for your help, i will try this method but i want to ask who can i know the more active sample?. Otherwise, can i apply this method to fractions with mixture compounds or is it for pure compounds only?.

You can try this method with mixtures as well as with pure compounds also. You can determine the IC50 values and compare the activity of samples using different concentrations.

Thanks for your interest. Can you explain the steps to calculate LC50 values for different samples with different concentration?

After you find the activity in your samples at a given concentration, make different concentrations of that samples ranging from 1 to 100 mcg/ml, as per your convenience (for eg. 10, 20, 40, 80, 100). Repeat the assay with these concentrations. You will get the readings for each individual concentration. Plot a graph and can easily calculate the IC50 value manually as well as using the regression analysis too.

Dear

A fast method for cytotoxicity is Brine Shrimp Test. Following two links may be helpful

http://www.sciencejournal.in/TBR-1-2-8.pdf

http://www.ijppr.com/PDF%20all%20edtions%20IJPPR/Vol5/Issue2/IJPPR,Vol5,Issue2,Article3.pdf

Thanks Mohamed for your help but the first link does not work can you send it again.

On other hand, my samples didn't dissolve in water, they dissolve in dichloromethane. So, what can i do in this case.

You can also search the term " Cytotoxicity test (Brine Shrimp Test)" in the Google scholar

Thanks for all but i want to ask what can i do if my sample don't dissolve in water completely??? What can i do with the Turbidity of the samples???? because some of these samples extracted from hexane and dichloromethane. I hope any one have a solution to this problem.

You may use DMSO to remove turbidity of the samples.

Can i dissolve all my fractions (non-polar and polar) in 2 ml DMSO as a stock solution and after that transfer from this stock 10, 100, 500 µl to separate tubes and complete them with 10 shrimps to 5 ml sea water????? what is the best methods to prepare the brine solution for hatching (i added 32 g of NaCl in 1 L dist. water is it OK)