Hi all, I'm a novice in this field of cellular actin restructuring...
I am treating RAW264.7 macrophages with a chemokine and hoping to demonstrate actin reorganization of the cell during subsequent migration.
Using Alexa fluor-phalloidin I have stained the actin of these cells at 5-15 minutes post treatment and observed a noticeable change in cellular shape (length and surface area) as expected.
I would like to relate these cytoplasmic changes to motility (ie if a cell has a certain actin-related features it is actively moving), so is to quantity the number of "motile" cells in a field.
I know this has been described many times before, but with each cell type having a different migratory phenotype, I can't seem to find an example of actin re-organisation in RAW264.7 macrophages which are migrating.
After looking at live cell time lapse of my cells (under random movement), I have determined that it seems these cells form Lamellipdium at the leading edge and contract toward this as they move. I also see filopodia at the leading edge, but also see it on most other sides of the cell.
I have looked to see if I can visualize membrane "ruffles" but am not 100% sure if what I am seeing is ruffling.
Can anyone help define the classical features of actin in a motile cell and how to identify them.
I have included an image as an example.
In the attached example my interpretation would be that the lower part of the cell is the leading edge with an apparent Lamellipodium, with some filipodia protruding.
In addition, would I be more prudent to use Peritoneal macrophages instead of a cell line. My aim is to address changes in migratory phenotypes in vitro at this time.
Sorry the Image doesn't appear to load into the question, I'll attempt to add it to comments. Excuse the resolution.
I second what Konrad said above. For lamellipodia, it would also help to stain for proteins such as cortactin. It should stain with a thin actin staining corresponding to lamellipodia at the leading edge. This assumes you are looking for classic 2D motility.
Since macrophages are random migratory cells, I would not recommend scratch assay.
Chamber assay is suitable only if chemokine act as attractant for macrophages...if chemokines just stimulate migration then it might not be helpful.
F-actin is another option if you specifically look for actin related changes.
Arp-2/3 activation assay is ideal if you look for actin growth cone formation at tips of filopodia or actin networks in lamellipodia.
If you have access to time lapse microscopy, that's the best to track migration of cells. There are tracking options associated with microscope's imaging softwares for this purpose. Since macrophages are constitutively migrating cells, you will need quantitative assays such as cell tracking in time lapse to show the chemokine induced difference in distance migrated and changes in directional migration.
Hi Jinesh,
This is exactly what we are planning to do. We hope to treat the cells with a stimulant, and see how they migrate following pre-incubation with other treatments. We have evidence that the cells migratory ability should be altered, and we hope to demonstrate this in a migration assay.
We have attempted to use ibidi chemotaxis slides but it seems the macrophages are not responding to even 200ng/ml CCL2. we only observe random migration. with slight direction.
In addition we are having trouble getting the software to visualize the cells against the background. I attempted to use calsein staining but i feel the cells might be dying from it. I am attempting Lenti-virus transduction of a GFP marker.
I wonder if you know whether adding LPS to the assay will get the cells moving. We don't care what makes the cells move, we could just like them to do it towards an attractant so we can calculate rate with or without our treatment.
The above question was aimed at seeing if we could define a motile phenotype using actin staining.
Try VEGF to induce migration. For stable staining without cytotoxicity, try PKH26..which can stay in cells for a week at least. It is safer than lentivirus..Good Luck..!!
Hi Adrian, you are in luck.
I completed my PhD at the University of Adelaide many years ago under the supervision of Leon Bignold (last check, he's still there but employed at IMVS now). He is an expert in leukocyte motility and associated shape changes (as I was for a while...refer early Harkin and Bignold papers. I can send pdfs if can't get). You should be able to grab a hard copy of my thesis in the library. If I was doing this work, I would attempt stimulation of cells in cell suspensions and fix the cells using 2.5% glutaraldehyde. This is a highly sensitive assay of shape changes related to chemotactic agents. See thesis for images of lamellipodia, uropodia, etc. Happy to chat anytime if needed. Contact me now at [email protected]
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