Our work is centered around the macrophage reservoir of HIV. We are trying to create resting macrophage cells in vitro so that they can mimick the resting cells in vivo. We have observed that non-coated plates are better suited for this purpose. Medium change is done once in 4 days. We face a problem of decrease in cell numbers with every medium change. Macrophages get detached and are washed away. Any suggestions from people working with macrophages would be of great help. Thanks.
Monocyte cell line used to generate macrophages - U9
Medium used - RPMI with 10%FBS, amino acids, insulin, sodium pyruvate and antibiotics
If you are trying to culture macrophages, you can't use non-coated plates. Once a macrophage is differentiated from monocytes to adhere to something, they usually only detach on their own when they die.
Why are you changing media at day 4? Differentation should take a maximum of 3 days. Once they are differentiated, (usually with PMA) you have to infect them or something. Without any stimulation, U937 die quite quickly once they are differentiated into macrophages.
THP-1 tends to do a little better.
If you are trying to proliferate the cell line, do it as a monocyte (suspension culture) not after differentiation.
Strictly speaking.. there is really no such thing as a "resting" monocyte derived macrophage in vivo, unless you are talking about tissue resident macrophages, but they are a completely different population and topic.
Dear, you will find useful information in links below:
Article Ionizing radiation modulates human macrophages towards a pro...
Article Macrophage-secreted factors enhance the in vitro expansion o...
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3570223/
I agree with Schen-An, ... there is really no resting monocyte derived macrophage in vivo. M-CSF and GM-CSF activate survival pathways in monocytes and facilitates macrophage differentiation.
When you change medium, don't change the complete amount, only change 1/2 the volume. We have seen macrophages do much better with this adjustment.
In our experience, peripheral blood derived macrophages did the best in IMDM + glucose and 10% human serum. We infected them with HIV-1 BaL and they lived for months - some 6 months with regular media changes.
Hi Gale, viruses have developed various strategies to protect infected cells from apoptosis, e.g. HIV-related proteins prolong macrophage survival through induction of Triggering receptor or binding to mitochondria thereby maintaining mitochondrial integrity. That's why your cells lived so long ... what happend without HIV infection?
We always had control cultures - T25 flasks - and as long as they had regular media changes, they survived as well. The HIV infected cells did metabolize the nutrients faster and required more media changes than the uninfected cells.
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