I work on MC3T3-E1 cell lines. The cells appear absolutely fine in flasks, but when I seed them in 12/24 well plates for mineralization (nunc thermofischer plates), then cells appear to die in few of the wells while being absolutely fine in others.
The morphology looks like as if after attachment, media was removed from the wells for cells to become dehydrate and die.
Please find the pictures attached.
Could you please try to help me to get possible explanation for this morphology?
Hi Heena,
Is there any pattern as to which wells are affected? For example are only the most perypherial wells afected?
Anna
Anna Wawer It is very complicated. If You have patience to go through this case history then please read on. :)
I am still trying to confirm the pattern. In a 12 well plate, we have 3 ROWS (A B and C) and four columns (1, 2 3 4).
Previously the wells used to get affected randomly (mostly the controls of my experiment)
but lately, only wells C1, C2 C3, C4 and B4 are getting affected.
Previously I thought that the is some problem is with the plate, as in may be the coating is not proper and even.
But then last time when I plated, by mistake I plated in a reverse manner. as in ROW A , I gave treatment that I used to give in row C and vice versa, Row B remaining the same.
But now also, A1, A2, A3 AND A4 and B1. Now because the plate was reversed, the treatment that I used to give in C1 was now in A4, C2 was now in A3, C3 in A2 and so on. This time, row A got affected. Also , I used plates from different batches of thermo and also from different companies. but problem remained same.
Now it is reasonable to say that may be the treatment that I am giving in these wells is a problem right?
But.................... my treatment is nothing but only Glucose in otherwise normal media. Moreover, I have a stock, say 10X. From this stock only, I give treatment in different wells in varying concentration, Which is not very high that cells cant bear it. Just one thing, in row A i generally give 1X, In B I give 2X and in Row C i give 3X. so I thought that may be there has been a problem with my stock itself, because when I am putting it in 3x then all the wells are getting destroyed. But what could be the problem??? I didn't put anything else in stock apart from Glucose.
Moreover, My few of my lab mates also faced the same problem, while their treatment was different then mine....though ,right now there wells are going fine, While few of them never faced such problem at all.
I even tried putting all sorts of chemicals that are available in lab (hexane, methane, formaldhyde, glycerol, diethylnylamine and what not...... in my plates myself to see if I get the similar morphology. ...but all in vain.
Ultimately I found out that when u starve the cells completely, as in remove all the media while the cells still attached to the plate, and leave it like that, then this morphology comes.
Which growth medium do you use (what is its glucose concentration?) and how much glucose do you add?
You mention you add concentrations the cells can bear, but can they really?
I suppose you also prepared fresh media and fresh glucose stocks and tried those already?
Your colleagues were using the same cell line and reagents when they also faced similar issues?
If I may some feedback and I might be mistaking on this one, but it seems to me you're using bright field on your "destroyed cells" photo and phase contrast for the "normal morphology" photo.
There's definitely a big difference between the two, for sure, but assessing the difference is harder when not using similar optics.
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