I have started working with primary human and murine macrophages and have successfully differentiated and used for infection experiments. I perform mostly CFU assays with bacteria. Although, I need to use them for other type of experiments as well.
The other experiments might involve Flow cytometry and imaging and I have two major concerns regarding these experiments:
Concern 1: For flow cytometry I would have to detach the macrophages from the surface of tissue culture wells and in doing so, I had tried trypsin but was not satisfied with the results ( Seriously sticky chaps ! ). I also found out that scraping using a rubber policeman or cell scraper might work although did not try yet. So I need some suggestions regarding how to detach them the best way.
Concern 2: For imaging, we usually culture cells on glass cover slips in tissue culture wells. Can we do this with Macrophages as well or there is a limitation in adherence to glass ? Please suggest
Looking forward to get some helpful tips.
Hi Sudip,
It is true that trypsin or cell scraper can work. However, I'm personally not a huge fan of those approaches with primary macrophages. The mechanical/chemical stress exerted onto the cells can be quite intense. In my hand, cell scraping always resulting in high cell death in th following hours, and trypsin tended to decrease the response of primary macrophages to pro-phagocytosis or pro-migration signals.
I prefer lifting my macrophages as follows:
- 1 or 2 rinces of PBS
- Incubation for a few min with PBS 10mM EDTA in the incubator. After this the cell should round up, and most of them should detach after gentle tapping of the flask.
- Collection of the cell suspension in PBS/EDTA. If necessary, pipet up and down to wash the culture surface.
- pellet the cells and resuspend in the desired medium.
Importantly, this works if your primary macrophages are cultivated on bacterial plastic (ie, non cell culture treated). Otherwise they get sticky as hell, and impossible to detach unless using very aggressive and cell damaging approaches.
I agree with Shadab: primary macrophages will adhere and spread on glass coverslip very easily. It will take a few hours in complete medium, or as short as 15min in serum free medium (if so, don't forget to replace with complete medium once the cells have attached).
Hi Das, following protocol might be helpful
1. Remove the media from the flask completely
2. Wash the flask with sterile PBS or HBSS and remove it completely. For cells that are difficult to trypsinise wash the cells with 0.05% EDTA(.05% EDTA in PBS) a couple of times before the PBS wash
3. Add trypsin to the flask or dish and incubate at 37 C for 5-10 minutes. Inspect the flask under the inverted microscope to look for cell “shrinkage” or detachment.
4. Gently tapping the flask and pipetting the medium over the cell monolayer.
5. Count the cells and adjust the concentration to be used for further analysis.
If u want to use cell scraper just gently tap the flask and scrap with scraper.
You can also use hypotonic solution instead of trypsin for disadhering your cells for flow cytometry.
Yes you can adhere macrophage on cover slip for imaging, it works
Cheers
Hi Sudip,
It is true that trypsin or cell scraper can work. However, I'm personally not a huge fan of those approaches with primary macrophages. The mechanical/chemical stress exerted onto the cells can be quite intense. In my hand, cell scraping always resulting in high cell death in th following hours, and trypsin tended to decrease the response of primary macrophages to pro-phagocytosis or pro-migration signals.
I prefer lifting my macrophages as follows:
- 1 or 2 rinces of PBS
- Incubation for a few min with PBS 10mM EDTA in the incubator. After this the cell should round up, and most of them should detach after gentle tapping of the flask.
- Collection of the cell suspension in PBS/EDTA. If necessary, pipet up and down to wash the culture surface.
- pellet the cells and resuspend in the desired medium.
Importantly, this works if your primary macrophages are cultivated on bacterial plastic (ie, non cell culture treated). Otherwise they get sticky as hell, and impossible to detach unless using very aggressive and cell damaging approaches.
I agree with Shadab: primary macrophages will adhere and spread on glass coverslip very easily. It will take a few hours in complete medium, or as short as 15min in serum free medium (if so, don't forget to replace with complete medium once the cells have attached).
To reiterate one of the points above, make sure to plate your primary macrophages on plastic petri dishes, NOT tissue culture plates. I've been able to readily detach primary macs with 0.25% trypsin or PBS/EDTA.
An alternative is to change medium for PBS and to put the dishes on ice for some time. The cells will detach.
Hey Sudip,
i am working with these cells. And for me scrapping after wash with PBS works very nice. But dont try with trypsin as it causes a lot of variation in result.
May be EDTA is a good idea, itried also but scraping gives me best results.
You may try and optimize with your FACS. I do the FACS in Gallios, Beckman-Coulter......
Best
S
Hello Sudip
This is going to sound crazy but I remember that speaking with other researchers they were talking about an antidepressant that promotes detachment of macrophages from tissue culture plates, right now I don't remember the name. Be careful with trypsin, it can damage cells.
Good luck
Jaime
Hi Sudip,
We work on macrophages only and we always use the same protocol which always works:
1-Wash with cold PBS and leave for a couple of minutes.
2- Use a cell scraper to scrape them.
Also, if you do this on ice, they will come off much more easily.
Regarding your second question, the answer is yes, you can use a glass cover slip in tissue culture plate. I have done this before and have never encountered any problems.
Good luck!
For FACS do not use cell scrappers. A lot of cells are damaged by this process. If you stain them with PI you will see a lot of PI+ cells.
Plate your macrophages in plastic but not tissue culture plates. This will allow you to wash them off easily with ice-cold PBS without calcium and magnesium.
Indeed, detaching these cells can be tough. I generally use the combination of Trypsinisation (till the cells round off) and scrapping together, gently, to minimise the effect of both.
Pedro, Sylvia and others, The idea of Ice cold PBS sounds good, but i was wondering that wont that again, be a stressor for the cells to later behave stressed/sightly activated? these cells are very sensitive to the environment, for obvious reasons, More so in the in-vitro conditions. I would like to know if the cells harboured this way work well in your experience. Thanks.
We work primarily with macrophages in vitro and do quite a bit of flow. We have found a lot of variability with trypsin, and prefer to wash the cells 2x with cold PBS+ 2mM EDTA, then place on ice for 30 mins in our FACS flow buffer+2mM EDTA to release the cells. This seems to work well for us, and results in the lowest background levels of death when stained with PI or a similar viability dye.
We have never tried the cell scraping method, as we typically work in 96-well plates.
Poojashri, I cannot answer for the others, but we seem to have little effects from the cold PBS as far as overall macrophage activation goes. However, most of our assays involve heavily activated macrophages, so any effects from the cold might not be apparent. That being said, for anywhere from 2-18hrs in our media-only, unstimulated macrophages, we typically see very little background death or activation when using our cold-wash method.
Hi Sudip,
I am wondering if your problem has been solved. I have been having this issue as well. From my work, I detached the cells but the cells were highly activated. So could you please let me know which method you use? Also, any activation exists?
Many thanks.
Dear Sudip
Try with Chlorpromazine. Even though it is an antipsychotic drug there is evidence that it detaches macrophages.
Good luck
For my experience, if cells are cultured too long, they finally attach very tight. I have tried both EDTA and scraping method, either only few cells become round or too much cells die in the following day. Maybe someone has a better way.
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