Are you using the same flasks as the other lab - do they coat them in any way? likewise are you using a the same source of FCS. Some MCF-7 media use insulin, pyruvate and estrogen. Does your media change colour - does it go yellow very quickly?Be careful not to over trypsinise your cells, spin down the trypsinised cells to remove the trypsin before replating. Sometimes replacing media too often can be detrimental as some cell lines produce autocrine growth factors. Try leaving a flask for a week to see if they re-attach. Check for mycoplasma infection if you can.

Ian

Dear Ian Teo

Thank you for the response

We are using corning flasks, which are similar with other laboratory with no further coating. The FBS we are using is from different source. Recently, I used serum without heat inactivation. Does FBS from different source creates such strong stress? if so, how long do you think it will take to recover?

About insulin, when I received them I added insulin in the medium recommended by ATCC, but cells went worse, after that I changed to DMEM/F-12 that used for the three cell lines in the other laboratory. However, very little change was seen. The medium color is orange and rarely changes yellowish.

About trypsinization, I incubate for about 7 minutes with little agitation, do you think it could be a reason? if so, how long do you think it will take to recover, duplicate and adhere to the flask?

I was changing medium every three or four days, after the cells got weak, I started changing every two days to ensure sufficient amount of nutrients.

Only T47D started reattach slowly with hardly noticeable increase in cell number after 4 days. Other two cell line are still non-adherent.

Non heat-inactivation will not get rid of complement in the serum, so you can have some toxic effects. Also very important is the speed and how long you spin down the cells. I found out that 200 g for 5' give you a fairly good pellet. I had trouble with other cell types (stem) when I spun them down at higher g or for longer time, they were all dead. But you can check very easily the cell viability by trypan blue inclusion, simply taking out a small aliquot of the media and adding trypan blue to them. Under the microscope, if they are blue, they are dead. Another possibility is mycoplasma contamination that makes the cells behave very weird. 7 minutes of trypsin plus agitation is way too much, simply remove the media from your flaks, rinse once with PBS,then add 2.5 ml of trypsin plus EDTA for a T75. Put in the incubator for 2' and they should be detached, otherwise tap the flask for 3-4 times against the palm of your hand. The cells will be floating by now. When you do cell count try to add trypan blue to the cell you are counting. If the cells are stained they are dead, so you can have a good idea about your cell viability

Hn,

I would stick to heat-inactivated serum unless you have reasons for not doing so. See if you can get the same batch number as your cell supplier. Extended trypsinisation and subsequent "failure" to inactivate trypsin can be detrimental - though usually serum neutralises trypsin. I would observe your cells under an inverted microscope until they start to detach and round up. Tap flasks to detach cells. Trypsinisation will remove cell-surface receptors / attachment factors which are necessary for attachment and subsequent growth. Frequent changes of flasks can also cause a slow attachment, as adherent cells often have to secrete extracellular matrix proteins / basement membrane proteins to ensure good adherence. If your media is not going very yellow after 2 days I would be inclined to leave it on the cells, or do a partial media change - cells do produce their own growth factors (you could try conditioned media from your original source if it is a nearby lab). As Danilo suggested you can do a quick trypan blue stain on your trypsinised cells. The presence of large numbers of dead cells in a flask will be detrimental to the living - can cause apoptotic death. You can get rid of the dead cells by ficolling. Mycoplasma can surprisingly both inhibit and "enhance" replication of cells. Ciprofloxacin treatment can "contain" mycoplasma infection - not eliminate it, but often slows down replication even further.

Persist, - it once took me nearly 2 weeks to get an expensive bought cell line to adhere.

Ian

Dear Ian and Danilo, thank you indeed for the valuable comments

I will go back to heat-inactivated serum, I usually pass the serum through 0.22um filter before use, I don't know if such practice affects protein contents of the serum?

Also, I usually spin the cells at 1500 rpm for 5 min, but I will decrease to the minimum level by which I can obtain the cells as pellet.

I stained the cells by trypan blue; the T47D are mainly living cells (over 90%), MDA231 fairly good (over 80%) and MCF-7 are the weakest and the variability around 65%.

I think I over trypsinized my cells, therefore, I will incubate them for maximum 3 minutes without agitation.

Is there an easy test to check for mycoplasma? can culturing the cells on agar show whether the cells are infected or no?

Thank you

https://www.researchgate.net/post/What_is_the_best_way_to_check_for_mycoplasma_in_cell_culture?_sg=2GcS8UHeGjinb8xNbEJPapWfjVxElH5wW3494DRTw2A2rTBD%2B%2FDrFvGkynQlCv%2Fe

Ian

Thank you again, I will modify my procedure and medium and get back to tell you the results.

Dear Hh, I would not worry about the filtration of the serum. After heat inactivation you will see a small flocculate precipitate in the bottle (complement's protein), but the amount of growth factor that you are going to lose during filtration is negligible, they will go trough the pores with no problem, in fact the pore size is not even able to stop viruses or mycoplasmas, just a little bit of protein could be stuck on the membrane due to electrostatic forces for example, and most of the protein in FBS is fetuin A with a smaller amount of BSA

Dear Hn, Mostly everything has been said. I have worked with MCF7 and 231 cells. Serum inactivation is very important. About adherence, these cells don't need coating, they're pretty good growing on plastic. And I usually performed spins of 5 min at 1200-1500 rpm. I think it might be a problem with trypsin. These cells need very short time of trypsin (30s-1 min) and rapid inactivation. You can kill them at that step. The other very possible explanation is mycoplasma contamination. Totally agree with the fact that they might grow faster or slower if they are contaminated (231 cells usually went faster in my hands). But the phenotype you describe is exactly what they looked like when I had a contamination, although usually the media got more yellow. We performed regular mycoplasma tests of supernatant by PCR. But you need to grow cells to confluence and then wait 2-3 days, collect the media and perform the test. If you are in a hurry and want to know it as fast as possible, you could grow some cells on a coverslip (just let them adhere) and then stain with DAPI. If they are very contaminated you should see lots of little blue spots all over the cytoplasm, similar to that: http://www.the-scientist.com/?articles.view/articleNo/19600/title/Cultural-Revolution--Mycoplasma-Testing-Kits-and-Services/

Thank you dear Naiara

referring of all of the above comments, it is likely that over trypsinisation injured the cells

As I was doing the same trypsinisation and passage procedure for all cells, in the case of T47D cells, the cells are adherent but the growth is very very slow, over a week now at 50% confluency. Do you think that because of trypsin? and if so, do you think they will recover fully?

Thank you

Dear All

Finally, the cells are growing happily.

With your comments and help, I could figure out the problem with cell lines. The problem was over trypsinisation and failure to inactivate trypsin. It seems that MCF and MDA231 are really sensitive to trypsin and may be injured if exposed more than two minutes.

Thank you