I am currently culturing THP-1 macrophages using 75 ng/ml PMA over the course of 120 hours. I have noticed that while the cells are attached to the well... I lose substantial cell numbers if the plate undergoes a wash step. I have tried coating the plate with type I collagen and it helps to improve the number of cells that remain but the cells are clustered around the edge of the well and refuse to grow in large numbers in the center. Has anyone else had this issue and how did you solve it?
Macrophages do not grow in the center of the well covered with collagen type 1, since they are not activated and they do not express collagen adhesion receptors -α2β1 integrin. along the edges of the hole, the visible layer of collagen is thinner or it is absent.
Article Adhesion of monocytes to type I collagen stimulates an APP-d...
THP-1 adhesion to collagen stimulated a tyrosine kinase-associated signaling response which included subsequent phosphorylation of p38 MAP kinase and increased association of APP with α2β1 integrin
I agree with Alexandr. In addition, do not grow THP1 cells beyond the density of 1 million cells per ml. THP1 also change properties if cultured continuously and show more clumping nature (being nonadherent cells, THP1 cells grow in clumps anyway, but bigger clumps are of concern and can be avoided this way). Use cells not beyond 10-15 passages and do not overgrow before use in experiments with PMA. With PMA addition, clumping should stop. If not, then probably the PMA is not working.
THP-1 cells treated with PMA are terminally differentiated macrophages. There is no scope for over growth and clumping unless too many cells are seeded in the very first place. Reasons for cells growing in the edges of the well could be either due to forced cell seeding or seeding the cells in less volumes of media into the wells. Moreover, growing for extended periods may result in cell death which may be one reason for cell loss.
Though THP-1 cells are semi-adherent, they become strongly adherent cells upon treatment with PMA. The level of adherence is so strong that needed either enzymatic treatment are physical disruption to get the cells off the surface. So, there is no need to use collagen to keep the cells adhered.
If these cell properties are not seen with the THP-1 cells you are using, then you may question the quantity of the cell culture
THP1 grows majorly on the corners in presence of PMA. I grow macrophage over the uncoated cell culture treated corning plate in presence of PMA. But 50 ng/Ml concentration of PMA (in my experiments) works above this concentration i found low viability.
Try to reduce the PMA concentration as it become toxic at high concentration. In my case i was able to gt macrophage in 100 hours only.
All the best.
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