I am trying to use RAW264.7 macrophages to observe migration. The ultimate aim is to treat these cells with compounds and see how they migrate.
At first however I wanted to just establish the assay, which means I simply want to see the cells migrate towards a chemoattractant.
I have tried a few different systems and cannot seem to get any migration towards the chemo attractant. I am currently using a Transwell assay, but still cannot seem to get the correct conditions.
This is what I have tried:
Cells at Passage 5-8, freshly brought up RAW264.7 cells, passages for 1-2 weeks post cryo.
Cells were grown to 80% confluence in DMEM 10% FCS, harvested by cell scraper in fresh DMEM,and resuspended in 0.5% FCS (lowest dose FCS I found these cells still happy at). Viability is at >95% on seeding.
Transwell assay used 8um pores on a 6.5mm membrane. Media containing 10% FCS was placed in the bottom well and the transwell lowered into this (At this point it seems no media leaks through).
Following this, ~40,000 cells were loaded into the top chamber in 100ul of 0.5% FCS media.
As a control, I had wells which had 0.5% FCS top and bottom chamber.
These were allowed either 6h or 18h to migrate after which, cells in the top chamber were wiped away using a cotton bud, and then rinsed with PBS.
After fixation and staining I saw no migration at 6h, and little cell migration at 18h.
I have also tried a real time xcelligence system (using FCS and/or 100ng/ml CCL2 as chemo attractants) and the ibidi live cell imaging system (again, with FCS and/or CCL2).
I know from actin stains that my cells are responding to FCS and CCL2 in a "migratory" manner, but I'm not seeing any migration.
Literature suggests I should see plenty in a very short amount of time.
I also noticed, that when both sides of the membrane is wet, it seems to leak media through rapidly when lowered back into a media. To me this suggests that a gradient cant hold?
What am I doing wrong? Is this cell line a dud at migrating?
Any and all help is appreciated.
I'm not working in this area but I think macrophages are differentiated from monocytes. It may sound stupid, but is it possible that macrophage does not react to the chemoattractants but monocyte does?
If your chamber is sealed properly media will not leak through the seals around the membrane however it will pass through an 8 micron pore size so you need to have your upper and lower chambers equilibrated (full and inject your chemoattractant into the appropriate chamber (usually the bottum chamber). I have always used the Boyden Chamber. I am not aware of RAW264.7 cells migrating into media enriched with FCS. Typically you need a chemoattractant. For example, C5a differentially stimulates the ERK1/2 and p38 MAPK phosphorylation through independent signaling pathways to induced chemotactic migration in RAW264.7 macrophages. This can easily be achieved using fresh or fresh frozen (complement present) bovine sera (if you choose) and injecting ~ 10 ul anti bovine IgG or any anti bovine serum antibody to kick of the classical complement pathway. You can also trigger the alternate complement cascade with LPS in a dose response fashion. If you are only using It does not look to me like you are using an appropriate chemotaxis stimulant. I would expect that the CCL2 ligand would stimulate chemotaxis of RAW264.7 cells but I have no data or seen any publications to support its use with this cell. I know LPS (10 ul dose response curve ) injected into in fresh bovine serum (10% bottom with 10% FCS on top in media of choice) works.
I have not worked with RAW264.7 cells before. But maybe the following suggestions can help:
1. Use a known/potent RAW264.7 chemoattractant for a positive control (i.e. LPS that Frank Bailey Gelder above suggested).
2. For many leukocyte cell types I have seen people using the 3 μm instead of the 8 μm pores. I wonder if the the 8 μm pores are too big and the cells can actually fell through. I have heard a few people reporting that most of their cells were at the bottom of their Transwell system at the end of the experiment.
3. As Frank Bailey Gelder mentioned, flow do occur through the 8 μm pores. You would want to have the system equilibrated (i.e. let the system sit for 15 min for the water height to equilibrate in the Transwell) before the introduction of the chemoattractant.
4. You would also want to use the right chemoattractant concentration. This could be tricky in the Transwell since it can be hard to control the right concentration at the membrane interface. Maybe someone have worked these minutia out (may have to dig the literature more). For example, 10 nM of fMLP on the bottom well most likely would have triggered the migration of neutrophil across the Transwell membrane since 10 nM is around the Kd of fMLP/fMLP receptor interaction for neutrophil.
Alrighty, hope your experiment pan out well!
In my experience the 8um pore size is far too large to maintain any gradient across the membrane. If that is the case, your cells will exhibit markers of 'migratory activation,' but you will not see actual migration across the membrane. Henry also makes a valid point: achieving the "sweet spot" of concentration will be a challenge. Too high of a concentration can result in pradoxical behavior. Best success!
Macrophages migrate very well towards chemoattractants, RAW cells included. I haven't had much experience with this cell line directly but I know they migrate towards CSF-1. I would suggest you look at protocols from Dianne Cox's collection e.g. Kheir et al., J Cell Sci 2005. We routinely use 8µM pore size for macrophage migration assays.
Dear Adrian,
I hope your experiments have been successful by now! If not, I would suggest that you do over night starvation of the cells prior to migration assay. We typically do 1%FCS and then use 10%FCS as a positive control even for this cell type and had no problems (4 hrs incubation is sufficient in our hands). I assume you are following protocol and coating the membrane with 1% gelatine? I would not worry about the gradient or pore size issues, but you will need to perform a range of concentrations of CCL2 in your case to get an accurate insight in their migratory response to this stimuli. In our case, we see nice dose-dependent increase in migration until reaching a plateau.
Good luck!
Hello Adrian,
Was the problem resolved? I will also be working on macrophage migration assay and I have no experience on it...From the literature, some people used 8um pore size, and some used 5 um, I do not know what to choose now.Could anybody here help me with it?
Thanks!
Yanrong
Hello Adrian,
Was the problem resolved? I ran into the same problem, may I have your suggestions?
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