Dear all,
I would like to see the effect of my drug on Raw264.7 polarization and I would like to use the Raw264.7 conditioned medium to treated cancer cells to see the anti-cancer effect. And I have the following detailed questions:
1. I plan to polarize these macrophage cells to M2 phage using murine IL-4 protein, and then treated with cells with my indicated drugs. I wonder if I have to change the medium and use this fresh medium as the conditioned medium to treated cancer cells? Since maybe some drug is still in the supernatant. And I wonder if I have to use the serum-free medium to do that to avoid some other variables?
2. Do I have to use a mouse cancer cell line to match the Raw264.7?
3. How can I compare the cancer cells in normal medium and conditioned medium. Since the macrophage cell is incubated in the conditioned medium for 24 h and the cancer cells will be seeded for 24 h as well, the depletion of serum or other nutrients will be almost the same. Therefore I can directly compare these two groups, right?
Thanks, everybody!!!
Hi, I suggest you have a look at transwells, which have two decks for compartmentalized co-culture. Some of the labs and your seniors at HKU Medicine should have plenty of experience using transwells.
A ref: https://www.frontiersin.org/articles/10.3389/fncel.2019.00230/full
RAW264.7 cells are, according to literature, more geared to the M1 subtype intrinsically.
It depends on whether you want to publish in a good journal in the end. To make a claim based on cell-based in vitro models, it is quite common to have multiple cell-lines of comparable nature to be used in the same experiments for comparison or validation. That is, you can have mouse macrophages, and use their conditioned medium to treat both mouse cancer cells (less convincing but usually easier to get desired results) and human cancer cells (supposedly more informative, but tend to have small effect sizes).
Beware that when doing your drug treatment experiments, the serum level should be adjusted (according to the right literature or after a good consultation with supervisors). 10% FBS for example may drown your drug effects or give rise to variability.
There is no such thing that all drug treatment experiments should proceed with a 24 h duration. Some preliminary kinetics study is recommended. For example, if you are interested in looking at transcript or cytokine expression, the time frames are definitely not that "standardized" at 24 h. Try to check and consult the good papers before finalize your experimental design.
Good luck!
Hi, I knew that the IL-4 has short half life, so I recommend to check the half life of your treatment...if it has short half life, it will be ok to use the same medium. Also, I do recommend using serum-free medium.
Your control group should also treated with conditioning medium too (From untreated macrophages M0).
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