1- yes cultured cells, A549 cell line (Human lung adenocarcinoma epithelial cell line)

2- yes 96 wells plate...

3- yes, 3 plates and each of them is for a day... they were set up in the same time and same passage of cells...

4- yes, I always look cells on the inverted microscope; before, I place the MTT dye to wells... yes, they are growing and proliferating...

I do standard MTT assay for understanding cell viability changes,

I set up the assay for 3 days (24 - 48 and 72 hours) in the same time and with same passage of cells.

first day; I sead 5000 cells/well

second day, I replace to sample + medium from medium

third day, I get ELISA result of 24 hours.

fourth day, I get ELISA result of 48 hours.

fifth day, I get ELISA result of 72 hours.

Dear Anja, thanks for your interesting.

my real problem is ELISA result, according to me there is no problem; however, my prof. said that if first day result is 0,400, secon day result must be 0,800 and third day result can be 1000-1250... because, second day or third day result must be double of first day. if this is not happened, we cant understand that cells are growing and proliferating...

I think that every cell line has a different cell cycle, so first day , second day and third day result can has near result, we must give importance to results of same plates. For example, if our control is 0,400, solvent control must be near to 0,400 and ineffective doses must be near to 0,400; if our results have this feature, we can accept the result is right.

Secondly, we must give importance to verification of sets. I repeat the MTT assay 3 times, if I get the same result every time. I think that the result is absulutely right.

I also get this information, MTT measures metabolic activity rather than cell number, so second and third day cells can have contact inhibition,so cell metabolic activity changes with degree of confluence MTT assay needs to be used with care with agents that cause cell loss.

shortly, I think that ever day result should evaluated alone...

what do you think about that??

The results are absolutely fine, yes is better if you analyse your data singularly. Also I want add that the data that you will be obtain from your blank or control, depends from which solvent you will use. In fact in my experiment last year I used DMSO that have a strong effect on cells because itis more cytotoxic, so I adjusted the concentrationto have a less effect on cells, also in my dilutionsamples.

1. Please ensure that the cells in the culture were synchronized by reducing serum concentration (i mean that they all were at the same stage of the cell cycle). Serum concentration should be less than 10%.

2. As you noticed here, MTT really measures cells metabolism, but does not necessary reflects exact cell number in the culture.

3. What is a doubling time of this cell line?

Dear Enzo; I use DMSO %0,3, it is ineffective...

Dear Anja,

the cells proliferatian is exponential... I do passage every 3 days and I divide to 4 flask from one. I set up an 3 days MTT assay and a flask cell line from a flask. For example, I have a flask A549 Passage number 33, I set up a 3 days MTT assay Passage34, and I have a flask A549 cell line passage 34. Namely, one cell give me 4 cells after 3 days ;))

Dear Natalia;

1. serum concentration is %10 FBS sigma and medium is RPMI-1640 Sigma (I always use same)

2. yes I agree with you, and I say that every plate should evaluated alone, they should not evaulated together, should they??

3. according to flasks; doubling time is less than 24 hours; because, in 72 hours one cell gives 4 cells...

thanks all of you for your interesting...

best regards

today, I set up a new MTT assay for same samples,and I changed something in the protocols, we'll see ;))

Dear Follower;

my new MTT assay results are compatible with old results, and my new results are like that ;

1. day control = 0,500-0,570

2.day control = 1.050 - 1.100

3. day control = 1.940 - 2.170

thanks for your interesting

Mehmet VAROL

ur results are absolutely fine. U will not get a definite fold increase in counts but it will show progressive increase in cell count and OD values. which u are already getting. so dont worry go ahead.

as it is a biological system, u cannot expect the exact fold increase everytime you perform an MTT assay over a 72 hour period and collecting data at 24 hour intervals in separate plates. But the control data u posted, says that your experimental procedure is correct; this is what actually we get when MTT assay is performed.

But one thing I want to inform you that while before adding MTT and before dissolving the precipitate, you need to remove the medium. So, please take care in both the steps so that while removing media, cells are not stripped off the cell layer in the 96-well plates which can give erroneous results in OD values of the duplicate/triplicate wells and may result in a higher standard deviation that will not be acceptable for your experiment.

Good luck with your next experiment...:)

Dear Mehmet,

Number of viable cells are directly related to DO on colorimetric MTT assays (please don´t confuse colorimetric determination with Enzyme-Linked Immunosorbent Assay (ELISA)), but in the same conditions. You might no get together lectures from different days because medium conditions, room temperature, pre-warm time of plate reader, etc. are differents ant they modify lectures. That is the reason why you need controls in all your plates.

If you have three time points, I suggest you to seed the cells in different times (0, 24, 48h) and read the plate to compare these results. In fact, I do it as I have told you.

I hope that I can help you.

Dear Follower;

first of all, thanks for your interesting and advices...

I observed some details with MTT assay for three years studiying in cell culture labrotory.

1-) I must use same brand materials for exact result (medium,flask, 96 well plates etc.)

2-) I must use cells after at least 3 passage from stock (same times 3 passage is not enough, so I must count cell every passage and I must draw a growth curve of cell line for understanding doubling time etc. and I must keep it same)

3-) I must solve the sample in the same condition ( I use hot water bath, for example 50 C 15 min.)

4-) I must do that ELISA results are read in same condition ( I use ELISA at 37 C 10 sec. mixing) because room condition is changing...

5-) medium must have same contion ( for example, I put in medium flask and the flask is stored 24 hours in incubator, after 24 hours I use the medium for cells)

6-) same cell line have pdl number and they are not immortal, I must observe them as morphological, proliferation time line etc.

7-) I must set up the assay for 3 time to understand the result are exactly same or there is a problem...

8-) I must keep everything under control in cell culture lab. so, I must observe everything everyday...

After all these things, if I think the assay has a problem, I set up new assay for 4. time, and I see there is not a problem ;)))

Thanks again everybody

Best regards

Dear Ramyar,

The problem was 2 years ago, however thank you for kind answer.