Dear all,
I've been working with the bone marrow-derived macrophages for a long time and now I am trying to culture the mouse macrophage cell line, the Raw264.7 cell line. I used the Gibco DMEM, high glucose, Glutamax medium supplemented with 10% FBS and 1% pen/strep.
I plated them in a 24-well plate around 50% confluence, and the second day (~12 hours), I stimulated them with M1/M2 cytokines, with the M0 wells as control. After another 12 hours, I checked these cells, and they looked wired to me in some M0 wells (see the attached image). At least 1/3 to 1/2 of those control wells (~5 out of 12 control wells) showed two different cell morphologies in one well (see the attached image). However, the M1/M2 polarized Raw cells look pretty consistent.
At first, I thought this might be caused by our old microscope, but today when I change the medium, I can even see a clear boundary between these two cell morphologies with my naked eyes (see the attached image).
Another thing I suspect is the 0.25% trypsin I used. Since this didn't happen when I used 0.05% trypsin. But my advisor said 4-5 mins of 0.05% trypsin is too long, and she suggested me using 0.25%. The digestion only takes 30 seconds to 1 minute with 0.25% trypsin.
I am wondering if any of you have encountered such a weird issue or am I missing anything when culturing Raw macrophages?
Thank you in advance!
- In general, For strongly adherent cell lines, trypsin of 2.5 % to 0.25% (10X to 1X power) is used. While the studies which require cell surface protein integrity, lower concentrated (0.05% trypsin) solutions are employed for 2-3 minutes in room temperature or 37 c for adherent cells . (Check this link) https://www.sigmaaldrich.com/technical-documents/articles/biology/cell-dissociation-with-trypsin.html
I think RAW not firmly attached, so 0.05% is enough.
- About the cell morphology: first you need to increase the cell density first before you evaluate the cell morphology, as the confluency is not reaching the satisfying percentage in the image. Taking in the consideration that RAW are polymorphic cells.
- Second, The small cells in the image, if not attached, may be dead cells or remnants of previous passage.
Dear Mohamed Hamed Abdelaziz, very informative, thank you!
Based on my cell culture experience, I feel like Raw cells attach to the plate stronger than most of the cell lines I cultured before, such as 293T and gastric cancer cell lines. It only takes 2 minutes for these cell lines to detach from the plate. However, the Raw cell line takes 4-5 minutes with 0.05% trypsin. I noticed before that 0.25% trypsin with the same incubation time as 0.05% causes severe damage to some cells, at least 293T cells. After that, I actually never use 0.25% trypsin.
The second thing you mentioned is the low cell density in these 24 wells makes it hard to discuss the morphology. This is very much true since when I plate more cells in these wells, I don't see two different cell morphologies. This happens only when the cell density is low. Today, when I checked the raw cells in the 6-well plate, designed for western blot, they reach the confluence with no issue and cells look the same, although with some dead cells. (see attached image).
You also mentioned these "smaller" cells might be dead. This might also be true since last night when I was collecting these cells from the 24-well plate, there are literally no cells in those "smaller cell" regions. It seems like they are all gone. But when I was counting cells before plating them into the 24-well plate, I used the countless 3 automatic cell counter, it says 95% cells are alive (the trypan blue method), which is a relatively high percentage. I don't know why they become dead in 12 hours in a 24-well plate. And they stay healthy in a 6-well plate.
Maybe I should they to seed more cells in these 24-wells?
Thank you!
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