# How to get enough cell alivefrom colon cancer tissue? - (Jan/23/2001 )

I am doing a experiment using MTT methed,but I can't get enough cell alive ,what I can get is a lot of dead cell,and most of the cell alive will be die after 3 days of culture. Please tell me the MTT method in details .Thank you very much.

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You have 2 choices. either you by the MTT SIGMA Kit which is really simple to use, or your lab is not rich enough and you have to do like that:

*prepare formazan

1-weight 5mg/ml of MTT ( pouder from sigma) using gloves and mask2-dissolve it in 25 ml of DMEM or your medium if different3-sterilise by filtering4-store at -20°C in black eppendorfs (MTT is Light degradable)

*Set up the multiwell plate

1-count celles in haemocytometer2-fill all the peripheric wells with sterile water3-fill the 1st column with medium only (It is the background)4-set up the plate with the right number of cells (it depends on your cells) add medium (100µl total max per well)5-incubate untill 80-100% confluence (40h)6-add 100 µl of MTT per well in the dark7-incubate 4 h8-add 100 µl DMSO9-read at least one hour after at 540 and 690 nm(ref)

*Calculations

1-do the mean for each column2-substract the mean of the backgroundcalculate the standard deviation and the standard error of the mean.

--

You have 2 choices. either you by the MTT SIGMA Kit which is really simple to use, or your lab is not rich enough and you have to do like that:

*prepare formazan

1-weight 5mg/ml of MTT ( pouder from sigma) using gloves and mask2-dissolve it in 25 ml of DMEM or your medium if different3-sterilise by filtering4-store at -20°C in black eppendorfs (MTT is Light degradable)

*Set up the multiwell plate

1-count celles in haemocytometer2-fill all the peripheric wells with sterile water3-fill the 1st column with medium only (It is the background)4-set up the plate with the right number of cells (it depends on your cells) add medium (100µl total max per well)5-incubate untill 80-100% confluence (40h)6-add 100 µl of MTT per well in the dark7-incubate 4 h8-add 100 µl DMSO9-read at least one hour after at 540 and 690 nm(ref)

*Calculations

1-do the mean for each column2-substract the mean of the backgroundcalculate the standard deviation and the standard error of the mean.

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