Dear all,
I have a very strange problem with my cells (HMLE cells with empty plasmid pBP). They are growing well in the flask, but when I seed them on a 6 or 96-well plate, they don't look very well. They don't divide and they look like a cell debris, I don't know how to explain it. Their pair, HMLE with Twist overexpression grows very well, I am using the same Humec medium.
What could be the problem? Could it be the mycoplasm? I will try the test, but if it was that, why they are growing well in the flask?
Thank you for your answers
Following hypothesis can be considered:
1) you use a low seeding density for the plates: 20,000 cells per well in 96-well plates and 300,000 for 6-well plates are usually appropriate seeding densities. However, it also depends on cell type.
2) The cell culture buffer is week, especially if its color changes.
3) If you have any contamination, the color of the medium changes usually very soon, even within less than a day.
4) maybe your transfected cells has been changed accidently with another cell line, which has similar morphology.
I hope these points can help you. Good luck
Hello, Marija!
Induction of an EMT in HMLEs by exposure to TGF-β1 resulted in the appearance of mesenchymal-looking cells (Figure 1A) and the acquisition of the CD44high/CD24low antigen phenotype (Figure 1C). These various observations showed that the HMLEs that have undergone an EMT develop antigenic markers associated with normal and neoplastic mammary stem cells. EMT process, we activated either Snail or Twist function reversibly in HMLE cells that expressed the respective transcription factors fused. HMLE-Snail-ER and HMLE-Twist-ER cells developed a mesenchymal morphology similar to HMLE cells stably expressing Snail or Twist
Article The Epithelial-Mesenchymal Transition Generates Cells with P...
TWIST promotes the transition of EMT (that is, to the phenotype of mesenchymal cells) and the expression of CD44high, which is an integrin and adhesion molecule. That's why when cells express TWIST they attach well.
Hey,
are your cells aggregating in clusters (if that is what you mean by debris). I don't have any specific experiences with the cell line you are using, but it is quite common that not all lines go well with all kinds of materials. Are your 96-well plates surface treated? What kind of polymers are they made of? For an easy fix, just try a surface treated, sterile well plate from another producer before engaging in any other testing.
Best
Sebastian
Cell culturing is complex, and it takes time to pick apart the process and isolate issues. Generally, the most common issues include abnormal growth patterns or spotty, uneven, or inconsistent cell attachment.
Check your 96 well plates they may not have been treated for tissue cell growth or they may be poorly treated.
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