I had subculture RAW 264.7 cell but at passage 8, the morphology of the cells were change. Is it because of the macrophage is already activated? What the different between the normal and activated macrophage? I use trypsin to detach the cell. Is it the concentration of trysin and the incubation time with trypsin will cause the macrophage to activate? What happen if we use the activated macrophage to induce with lipopolysaccharides in our study?
You can check for the expression of macrophage activation markers like CD80 and CD86. You can also check whether TNF-a is being produced or not. Trypsin treatment normally doesn't activate the macrophages. If you use activated macrophages for LPS stimulation, you will not see much effect of LPS as the cell is already activated.
Hi, The cheapest assay for determining if cells are activated is to monitor nitric oxide using direct Griess assay in culture supernatant. Activated cells secrete high levels of nitric oxide, while cells not activated do not synthesise this. Remember you must use DMEM culture medium. RPMI1640 results in high backgrounds. You can then go to the more expensive biomarker assays like CDs and cytokines.
If your trypsin does not contain pyrogens, the cells will not be activated. Agree with Niladri that activated cells already synthesise biomarkers of inflammation, so effects of LPS will be masked due to high backgrounds.
So what the cause of macrophage being activated or change their morphology?
For activation it can be due to a number of factors: Did you get a new batch of medium, serum or antibiotics? If so check if these components are pyrogen free. This can be done by LAL assay. For the morphology change: This can be due to activation or mycoplasma infection of the cells. Could also be due to other factors. It is best to get a new batch of cells and get pyrogen free medium components before you start.
Mycoplasma contamination is another possibility.
The cheapest way to fix your problem is to throw out your cultures, media, and other reagents and start a fresh culture in IMDM + 10% serum + other supplements.
If you really do a forensic analysis of your macrophage activation, it is going to cost you $$ and time. Flow cytometric analysis of CD80, CD86 and intracellular expression of TNFa, IL-1b, IL-6 can be done in one expt.
1. Stain for CD80, CD86 and then fix the cells
2. Permeabilize using BD kit for this purpose
3. Intracellular cytokine staining for TNFa, IL-1b.
TNFa will tell you about TLR-mediated cytokine activation
IL-1b activation will tell you about inflammasome mediated activation.
As I said before, this experiment will cost you money and your valuable time. None of your findings are publishable. Go for it, if you have time.
Yes its better if you start afresh rather than measuring whether macrophages are activated or not. If you have made cryofrozen aliquots of the cell line, revive them in fresh batch of media/serum. If these cells have been stably transfected with a bacterial/viral gene, that can also result in activation.
I am working with RAWs for years and did virtually everything with them. They grow so called semi-adherent, so some of them are loosely attached, some of them spread nicely and some of them are round. Best is, when you see nicely spread cells. Wrong culturing conditions (wrong splitting ratio, too overgrown, too high split, not singularized properly before splitting) changes their morphology and they tend to grow in circles and batches and they are not that spread anymore. In what way did the morphology change actually?
Activation of those cells is super easy to determine: They get more adherent and the diffraction patter changes, they seem to get flatter. A bit like a sunny-side-up. But, naturally, if all your cells are activated you will not see that easily. Same goes for many other things ... without a good negative control, it can be hard to see.
As said before, start with a new vial AFTER you determined whether you got a Mycoplasma infection ... this can be nasty. All other infections you should anyways see.
Visible observation: The morphology of cells changes i.e. after activation they develop epithelial cell like structure.
Also cells will take much time during trypsinization.
Biochem solution- checking NO level will help
Marker based study can be done but its costly
gud luk
I absolutely agree with Ahmed, trypsin is not the first choice for detaching RAW264.7. You should strictly follow the original protocol which use scraping for detaching cells. Another important issue is that you should not use 100% fresh medium after passaging. You should always add some of the old medium. If required, I can send you our detailed protocol.
However, if cells look like you have described, you should discard the cells and you should start a new culture from your original stock.
Dear Dr Stefan, thanks for your response. If possible, may I get the detailed protocol? Your help is really appreciated. Thanks.
Thank you all for fruitful information.Regarding RAW264.7 cell line, could any one help me to know what will happened if i Co-culture RAW264.7 macrophage with bacteria for 1hr at 37C without CO2 line?
does any body tried to incubate these cells in that conditions?
Dear Muna,
without CO2 for a short time, nothing will really happen. Add HEPES to your media to provide buffering, as the CO2 is used as a buffer in the end. What will happen is, that your RAWs will be heavily activated and start producing Tnf, Il6, Il1b, ROS etc. and they will phagocytose the bacteria.
Good luck!
If you are using DMEM media for your RAW cells, I recommend 5 mM HEPES following Florian's recommendation. That's what I used and it is quite enough.
Good luck!
Macrophages are highly sensitive to external stimuli. Even trypsin can activate these cells.
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