Sunday, October 07, 2007

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)

10 comments:

The Lab Freaks said...

Najib!

Think u write the volume wrongly..supposed to be microlitres rite? U wrote ml instead.
Why do u have to add in cell lysis reagent? For my MTT assay, i dun remember adding in cell lysis reagent.ALso, whats the different concentration that u use for the drugs?

Sry for the so many qns..haha..cos i was doing MTT assay also..

Charmaine
TG01

MedBankers said...

Hi Najib, may i know whats the purpose of adding in trypsin?

Thanks.

Pei shan
tg02

MedBankers said...

hey

WOOO!! sound like mammalian cell technology.... IS the procedure same as what we learn during lessons?

do you count the cells using Kova chamber (with the 90grid) before u dilute the cells line?

elaine

Kent said...

Heyy,

Interesting MP title u have there, but its gonna be challenging too.

A few qns, what is the unit of formazan production? How exactly do u calculate the cell viability from the amount of purple formazan produced?

Thanks!
Kent
TG01

The Lab Freaks said...

hey najib! (:

haha the MTT assay seemed farmiliar like wad we did during MCT session! but anws, wads the function of the cis-Platinum(II) diammine dichloride? what kinda drug issit?
and you say "the reduction process of formazan takes place only when mitochondrial reductase enzymes are active".. so whats the optimal temp? our body temp?

Thanks! (:

Natalie

The Lab Freaks said...

Hi Najib,

To be honest, I can hardly understand ur post. Can I ask, how is any of this related to the subjects we are taking this sem? heh.

Okaylah, also want to ask. how does finding out the cell proliferation directly contribute to ur research? =)

Sharifah
TG01

The Lab Freaks said...

Hi Najib!

I noticed that the cell conc that you used is quite little too. 1 X 10^4 cells / 100uL means its like 100cells/1uL only?

Royston Tan
TG01

The Lab Freaks said...

OOps Najib..I realised that its not ur fault with the volume..haha.its the school com's prob..

Charmaine
TG01

The Lab Freaks said...

Hello guys sorry for the late replys.

To Charmaine..
HAhahhah the sch com didnt spoil. I change it when i got your comment. The lysis reagent is to stop and activity of the cell towards the MTT reagent. With this all wells stop their reactions at the same time and thus helps to give accurate results.

The drug concentrations are 0uM, 0.3125uM, 0.625uM, 1.25uM, 2.5uM, 5uM, 10uM and 20uM.


To Pei Shan. This is the same question that Shu Hui asked me in my previous post. OHhh you never read ah!! Pls refer to my previous que and ans to get your ans.

To elaine
Another one who never read my previous post ah. TSk TSk TSk. Pls refer to my previous post for your ans. There are slight differences in the techniques which are all covered in my previous posting!

I never heard about the Kova chamber. Hope you can explain to me what is that chamber all about. Thanks. By the way i use the haemocytometer to count before diluting my cells.

To kent.
Im not sure about the unit of formazan production. But from what i know, i calculate the absorbance of the assays using a multi plate reader at 570nm. With the absorbance, i will plot a graph.

Y axis..... absorbance,
X axis..... drug concentration.

With that i will find the IC50 of the drug.

To Natalie
The function of cisplatin is an inorganic platinum agent (cis-diamminedichloroplatinum) with antineoplastic activity. Cisplatin forms highly reactive, charged, platinum complexes which bind to nucleophilic groups such as GC-rich sites in DNA, inducing intrastrand and interstrand DNA cross-links, as well as DNA-protein cross-links. These cross-links result in apoptosis and cell growth inhibition.

The optimal temperature is 37 degrees celsius.

To Sharifah for the wonderful question.

With the proliferation assay, i am able to find the IC 50 of the drug(taught in bpharm). With the IC 50 value of the durg, i will see if there is any realtions with the telomere length of the cells. Details will be in my report.

To royston
Ohh i was taught to use that concentration. Maybe different experiments have different cell concentrations.

DONE!

Unknown said...

nice posting.Single and two layer Orbital Shaking Incubator systems are one kind of designer construction. These Incubators are available with a choice of heated and refrigerates models.

Shaking Incubator Manufacturers