Problem in harvesting with MTT - (Apr/14/2011 )

Hi all,

I am facing problem in harvesting the assay with MTT (5mg/ml).

My protocol is:-
Inoculate 100ul of cells at a density of 5 X 105 cells/ml.
Incubate for 24 hrs for attachment.
Discard media and then add 100ul compound with diff conc.
Again incubate for 24 hrs.
Then remove compound and add 10ul MTT and incubate for 4 hrs.
Centrifuge at 125g for 10 mins.
Remove MTT and add 100ul of DMSO to dissolve formazon crystal.

I am facing problem while removing MTT some of the formazon also come out in pipette n m not getting proper result. Plz help if you hv any other method of harvesting.

Thanx.

enthusiastic on Thu Apr 14 09:41:30 2011 said:

in my case, I dont centrifuge and remove MTT. after the addition of MTT and 4h of incubation, I just add DMSO and wait 15 mins then get the OD value. it works well.
BTW, I never heard a method to remove MTT. Good luck

gyma on Fri Apr 15 17:38:21 2011 said:

sorry I made a mistake. I didnot use DMSO to dissolve formazan, instead I used MTT lysis buffer composed of 2-propanol(500ml), triton-x(20ml) and 36%HCL(2ml). So I dont need to remove MTT or medium.
good luck.

gyma on Wed Apr 20 07:12:06 2011 said:

Hi gyma,

Thanks for your kind reply. I will definately try this.

I just wanted to know do you prepare the MTT lysis buffer with the above mentioned composition only? and at what wavelength you take absorbance?

Awaiting for your reply.

Enthusiastic

Dear Enthusiastic,
In my case, I seed the cell for 24 hours same as of yours and then generally add the drugs or compounds of interest in the same system. After 24 hours, I directly add 20 ul of MTT ( 5mg/ml ) in the same system and leave for 3-4 hours. After that, I remove the conditioned medium using suction diligently and dry my anchored cells for about 10 mins followed by dissolution in DMSO directly and then OD is taken at 595 nm. This simple procedure eventually works for me every time.
P.S. You need to show kindness to your cells. If that doesn't work, why don't you try some other methods like CKK-8 method where you dont need to remove the supernantant as in case of MTT.

Hi gyma,

I tried your method of harvesting but I didnt get proper result, yellow colur of MTT did nt disappear completely so result was not proper.

The difeerence was I used 35% HCl as 36% was nt available and Triton X was Triton X-100 will it affect the result?

I wanted to know how much amt of MTT lysis buffer u add in wells? Do u incubate the plate? if yes then what is the period of incubation? Do u incubate it in dark?

Can u plz give me ur protocol in detail?

Thanx.

Hi shymadi,

Thanks for reply. Can u plz tell me what is suction diligently becoz i remove the test compound with micropipette only.

I do the procedure as below, I am getting result always,
first i seed the cells then i will treat the cells by different dilutions of drug from 100 to 1.5ug/ml, this is all in 96 well plare, and triplicate, after incubation time (24,48 and 72) i will not remove any media, depend on media availble in each well i will add 10ul of mtt solution to each 100ul of media in each well, then after 4hrs i will remove the complete media by taking the 96wel in 45" angle, remove the media carefully, the tips should not touch bottom of the wells, then add 100ul of DMSO, read in 570nm.
If your cells are detaching from the bottom when you are removing the media, it may because of the plate which is not good quality. or it may because of the cell line you are using...