Hello,
I was wondering why there might be cloudiness in THP-1 suspension cell cultures. The flasks turned relatively opaque after about two days of thawing, and are pictured below at five days after thawing. When the cells were cultured in DMEM media containing one percent penicillin-streptomycin (second picture) the media looked much less cloudy.
Contamination by a microorganism has been considered, but signs of contamination are not visible under the microscope. Also, the recommended media for THP-1 is RPMI without antibiotics, so I am unsure if the DMEM media is clearer simply because less cells are growing. Could the cloudiness be the result of the cells being high density? If the cells might be contaminated, what should we be looking for under the microscope to verify?
Thank you all for your help.
Hi Ritvik,
Cloudiness is a typical sign of bacterial contamination, however in THP1 due to its non-adherent nature has to the naked eye always the appearance of a cloudy medium, especially when high density is achieved (follow provider instructions, THP1 are very sensitive to it). So my recommendation is to do the passage (lowering the number of cells), culture the cells without antibiotics or antibiotics to observe any cryptic contamination, and ideally culture a few microliters of the suspicious medium into agar plates (suitable for general bacteria growth) and observe in few days whether some colonies appear.
A change in the color of the medium, the disappearance of its transparency, and the appearance of cloudiness in it is evidence of bacterial contamination.
Examine the medium microscopically (40X) for the presence of motile organisms.
If you do not find bacteria, it may be contamination with Mycoplasma, which is too small to be seen with an ordinary microscope.
Grow the cells again and try using gentamicin with penicillin and streptomycin.
Use 0.22 micron filters when preparing the medium
https://www.researchgate.net/topic/THP-1,this link might be very useful.
THP-1 cells are non-adherent, so all treatments you perform to promote their attachment are going to have an impact on them.
https://www.researchgate.net › post
How to prepare adherence cell line from THP-1 suspension cell line ?
THP-1 cells were differentiated into macrophage-like cells (THP-1 macrophages) by incubation in the presence of PMA, which leads to a macrophage-like phenotype characterized by changes in morphology and increased cell surface expression of CD11 and CD14 [10,11,14,17,21].Mar 14, 2018
https://journals.plos.org › article › jo...
The phorbol 12-myristate-13-acetate differentiation protocol is critical to ...
https://www.researchgate.net/post/Why_do_THP-1_cells_adhere,kindly check this link.
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