Cell lines and MTT assay

Eloooooooooooo. This week i am going to share with you guys about the cell lines that i am using for my project. For all the cell lines, i had to perform MTT assay aka cell proliferation assay. I will further explain what MTT assay is later in the post.

Firstly lets talk about the cell lines. For my project, I am using 8 human cancer cell lines and they are:

1. A431 (skin cancer)
2. HepG2 (liver cancer)
3. Chang Liver (liver cancer)
4. A427 (lung cancer)
5. CRL-2119 (pancreas cancer)
6. MDA-MB-231 (breast cancer)
7. CCL-244 (colon cancer)
8. CCL-220.1 (colon cancer)

To learn the full profile of the cancer cell lines and also the difference between the same cancer (eg. the 2 types of colon cancer), go to http://www.atcc.org/.

Based on the recommended medium (from atcc.org) ,

A431 is grown in DMEM (Dulbecco's modified Eagle's medium)

HepG2, Chang liver and A427 are grown in MEM (minimal essential media)

CRL-2119 and MDA-MB-231 are grown in a 1:1 mixture of DMEM and Ham's F12 medium

CCL-244 and CCL-220.1 are grown in ATCC-formulated RPMI-1640 Medium

Now I will talk about MTT assay.

MTT assay is a laboratory test and a standard colourmetric assay for measuring cellular proliferation. The assay measures the changes in colour and it is used to determine cytotoxicity of potential medicinal agents such as drugs.

Method to perform MTT assay(assay is done in 4 days)

1. Discard spent media from culture flask into waste bottle
2. Wash with 10mL PBS and discard
3. Add 2ml of trypsin and incubate for 2- 3 minutes
4. After cells are detached, add 8mL of media to deactivate the trypsin and mix well
5. Transfer 10mL of cell suspension into a 50mL centrifuge tube
6. Centrifuge the cell suspension at 1500 rpm for 3 minutes
7. Discard supernatant and resuspend cell pellet while washing with 10mL PBS
8. Centrifuge at 1500 rpm for 3 minutes and discard supernatant
9. Resuspend the cells in 5mL-10mL of fresh medium
10. Transfer 50mL of cell suspension into an eppendorf tube and add 50mL of trypan blue
11. Add 20uL of the stained cells to a hemocytometer and perform a cell count
12. Prepare cells to obtain a final no. of 1 X 10^4 cells / 100uL by dilution if necessary
13. Seed 1 X 10^4 cells into each well of the 96 well plate 9 (groups of 5)
14. Incubate overnight at 370C, 5% CO2 incubator
15. Next day, add 1uL of drug* into each well
16. Incubate overnight at 370C, 5% CO2 incubator
17. Next day, add 25uL of MTT reagent into each well
18. Incubate for 2 hours at 370C, 5% CO2 incubator
19. After 2 hours, add 100uL of cell lysis buffer into each well
20. Incubate overnight at 370C, 5% CO2 incubator
21. Next day, obtain the results from multiplate reader (absorbance at 570nm)

*Drug used is cis-Platinum(II) diammine dichloride of different concentrations

The yellow MTT reagent is reduced to purple formazan in the mitochondria of living cells. Then a solubilization solution (cell lysis buffer) is added to dissolve the insoluble formazan product into a coloured solution. The reduction process of formazan takes place only when mitochondrial reductase enzymes are active and therefore conversion is directly related to the number of viable cells. When the amount of purple formazan produced by cells treated with an agent(drug) is compared to the amount of formazan produced by untreated control cells, the effectiveness of the agent in causing death of the cells can be deduced through the production of a dose response curve.

Ill end here for now.

Najib Bin Hamid (0503217B)