I think it really depends on what you want to use them for. For example cell lines like 293T / HeLa we use normally for protein overexpression/interaction studies. For this we dont bother about passage numbers. But if u are using epithelial cells and studying some parameters like invasiveness, tight junctions, channels etc etc. long passage cells are finally no more epithelial- functionally /morphologically. Then u could be in trouble.

I think it really depends on what you want to use them for. For example cell lines like 293T / HeLa we use normally for protein overexpression/interaction studies. For this we dont bother about passage numbers. But if u are using epithelial cells and studying some parameters like invasiveness, tight junctions, channels etc etc. long passage cells are finally no more epithelial- functionally /morphologically. Then u could be in trouble.

If you are doing expression studies, Then you need to be sure about passage, The expression of several membrane protein changes with higher passage number and in several paper they mention it also. if with MTT you are just evaluating some chemical insult and recovery effect of some drug then the cell passage wont be a big matter of concern unless the cells are in good condition.

yes, i will use THP1 or HepG2 cells for my expression studies (pcDNA3.1+Insert+GFP)

In general, I never see passage numbers of cell lines added to the materials section of research articles. However, you should keep track of passages when performing experiments, specifically using cancer cell lines because of genomic alterations and clonal selection that can involuntarily be acquired after several rounds of splitting.

Cells may behave differently when at passage 70 than at e.g. passage 7, both regarding proliferation (doubling time) and functionally (transfection efficiencies, migration, invasion). For example, we have noticed that MDA-MB-231 cells proliferate more intensely at later passages when compared to early passages. So proliferation assays may turn out not to be reproducible when performing on highly different passage numbers.

For immortalized cell lines (like A549 and MCF7), I believe subculturing until passage 50 is considered acceptable, but when performing experiments in biological multiplicates, keep passage numbers in close range between different experiments.

I think unless you are using primary cells, passage numbers are largely unimportant, because they are very likely to be inaccurate. (ie. how can you say how many passage a tumor-derrived cell line has undergone before it arrived in your hands/lab?)

With primary cells passage numbers are a must though.

@Nicholas Wallace: you have a point, but if you obtain them through an official channel like ATCC, ECACC, ... the provided cell line information should be accurate.

Some reviewers might ask you about passage number during the review process, but it's pretty rare that exact passage numbers are mentioned explicitly in the final manuscript.

If a reviewer has a question about passage number, I usually tell them the maximum passage number I used in my studies. Personally, I've never used passages past 30 (after receiving them) and always try to freeze back excess cells from the first 3 passages after receipt so we have enough early passage cells to recover after I discard older cells. I would at least mention where you got the cells in the manuscript, especially if you received them from another lab (not all labs keep track of passage numbers)

As Lijo and Ridha mentioned, clonal selection due to artificial lab selection pressures can occur, especially in cancer cell lines. Early generations of MCF-7 cells attach loosely to tissue culture surfaces, but standard passaging and trypsinization usually selects for the more adherent populations. If you look at publications that use MCF-7 cells, MCF-7 can look like anything from spherical and clumpy to flat and fibroblast-like with tight borders. The latter morphology probably indicates an MCF-7 cell line that has been passaged much more times due to its more adherent phenotype.

Since the MTT assay relies on NADPH metabolism, if there are any changes in NADPH reductase activity with increasing passage number, then it might affect your results.

It depends on what you are working with, not essentially limited to the question of publication.

Cancer cell lines- clonal selection might be a problem, but i would say this problem is really common with cancer cell lines going from lab to lab. Usually I avoid using passages over 30 at all. I also freeze cells from the first few passages in order to keep the passage number low.

Immortalized cells, which I am working on currently are more sensible. We can see alterations in cell morphology over prolonged culture, also the response to cetrain stimuli is different, so I tend to use cells from the same passage (+/- 2 passages) for my experiments. After passage 25 we usually discard them.

Primary cells- here also the passage plays an immportant role, as well as in some cases donor age for instance. Here passage number and consistency over subsequent experiments is a must. Since you have so many influencing factors and donor variability, at least passage number is something that you can control.

Irrespective of its' importance for publication, I wouldn't use any cell line beyond 2-3 weeks (with the cell lines you have mentioned, not more than 10 passages). Best way is to freeze as many vials as possible initially and pick a fresh one every 3rd week; this will reduce the variability between your functional assays (cell proliferation assay in your case).

Thank you very much for all your guidelines

8-10 passages should be okay Senthil, beyond which we generally do not go.!

Yes, passage number is important. It not only shows your detailed recording procedure but also important to claim your research if there is any dispute.

As far as maintaining the passage number, it depends on what purpose you are going to use the cells. For example if you are using say MDA-MB231 for GAPDH as control then even 100 passage is good however, if you are doing any important aspect of your research then lower the passage number, the better it is.