Do you have a positive control for the migration assay working? What is your membrane pore size?

The speed of settling of the cells in the well shouldn't be a problem for migration, but it could affect the homogeneous distribution of cells and afterwards the quantification of the assay. If the cells have migratory skills they should be able to squeeze through the pore and attache to the bottom membrane. A positive control will help to elucidate if the assay is being performed correctly.

The coral cells are about 10uM, the pores are 3uM, the cells make a lot of mucus so I've most recently added e/z which was better but the cells are forming a visible layer on the transwell membrane (100,000 cells 12 well plate transwell, 200uL top volume, 1mL media with PAMP allowing 2 hours for migration) within 5-10 min. I feel it's more a settlement blockage issue than attractant, I have been doing small trials with LPS and peptidoglycan, as we have no "known positive control". I'm thinking a PAMP or bacteria infused disc permeating/ diffusing into semi solid agarose would be a nice way to determine active migration as long as you could quantitate the cells.

Most of my experience is in migration assays for cancer cells, so probably the assays in coral cells are run in a different way . I'm sorry I cannot help, but at least I can guess that the pore size might be right as 3uM is use for Neutrophils that are around 8-9 uM.

It really will depend on the properties of your cells. If they are mucin producing epitheial like cells, increasing the pore size to 8 microns (normally used for epithelial and fibroblast cells) may help (3µm may be works good for highly migratory and squeezing cells like neutrophils). Do you coat the membranes with something?? If not, it could be tried (coating with some protein). This may prevent your sticky cells from directly binding the membrane and sticking around and could make them more motile over the membrane. Good Luck

You can try using a Dunn chamber for cell migration. The cells need to adhere to a glass coverslip. Also, the agarose/agar plug containing attractant could work. This works for Dictyostelium discoideum cells and they are about 15-20um. These cells do not work very well in transwells either. I have attached two papers that have alternative methods for cell migration assays. I personally avoid transwell assays since the cells cannot be imaged in real time and chemotaxis and gradient parameters cannot be accurately measured.

Paper with an some alternative migration assays. This may be more than what you need for your experiments.

Article On-Chip Open Microfluidic Devices for Chemotaxis Studies

Can you use an adherent cell assay? We routinely use an assay in which cells are plated onto plastic culture dishes or multi-well plates that have a small silicone rubber mask (sylgard 184, available via Amazon.com) stuck onto the bottom. The cells are plated and grown to confluence, the mask is then removed to create a cell-free zone that can be monitored for migration. Attached is a description of the method as we use it (we didn't invent the idea, we just adapted it to our needs). Its cheap and easy, and you can make the masks in whatever shape or size works best for you. It also doesn't induce cell damage the way that a scratch assay does.

Two important things to consider: 1) cell proliferation. Proliferating cells will happily fill a cell free zone but don't really represent migration. Use of mitomycin C or some other mitotic inhibitor may be necessary to distinguish genuine migration from proliferation. 2) Chemotaxis. The mask assay does not have an inherent gradient of a chemotactic assay like a Boyden chamber assay does. If you need to assess chemotaxis specifically, then you would need to provide a source of chemotactic agent. If you want to see a general activation of migration the agent could be added to the medium. If you need to test directional migration, a localized source of the chemotactic agent would be needed. Theveneau et al., 2010 presents some good ideas on how that might be done.

Since you are thinking about an agarose-based assay, would this publication help?

http://www.biotechniques.com/BiotechniquesJournal/2007/October/Improved-agarose-gel-assay-for-quantification-of-growth-factor-induced-cell-motility/biotechniques-43304.html

You might need to contact the authors and ask for a glass mold. I have no idea about your cells so I can't help more.

Hi Kelly-Anne,

My experience with migration is usually with smooth muscle cells. What type of cells are you using? have you ever tried Boyden chamber method? It is very easy to perform once you get used to it. Good for any type of cells. You can have a max of 24 different conditions per run (given that you have a douplicate for each condition at least).

I hope this is helpful and please let me know if you need more help.

Good luck,

Laila

Try using a larger pore size.

If reduce the cell density will work or not? To my opinion your cells are too crowdy.

If you are using agar as a matrix instead of a cell layer it may clog the pores, no matter what the size. Dilute is as much as possible.

For an agar matrix, the pores would not be an option, would need to do a diffusion gradient style assay. I think the coral cells need more friction to move, hence the agarose/ collagen/ lysine binding idea. The cells do not bind well to regular TC plastic, have tried a few free fancy BD plates also reduced serum but no better.

I could try larger pores but having a lot of experience with mammalian cells in this assay worry a larger pore will mean more non-specific movement and free zooxanthellae could "fall" through.

Cell density could be reduced but at the expense of accuracy, but I don't think that's the issue.

You might be able to adapt a Dunn chamber (suggested above by Gus Wright) for your use by pre-coating the glass coverslip with poly lysine, concanavilin A, gelatin or some other matrix that your coral cells will adhere to, then plating the cells and doing the migration assay using the Dunn chamber. Should be no pores to clog that way and the proper matrix should give you the adherence you need.

A totally unrelated comment: My family just got back from spending about 10 days in Queensland. The barrier reef and its coral formations are stunning. My daughters wanted to smuggle a wallaby home. Wonderful people, too. Good luck with your studies.

Dave Sherry

You might like to try the µ-Slide Chemotaxis Assay provided by Ibidi. http://ibidi.com/applications/chemotaxis/u-slide-chemotaxis-3d/

It works really nice but it takes sometime to fix the right parameters for your system. Good luck!

There are more techniques and tricks to overcome the problem you described.

1. The simplest way is to use a relativeley low cell density - in this case there is no confluent layer on the bottom of the transwell, identical cells have the chance to reach the pores. For sure in this case the evaluation is not so easy (e.g. light microscopy of sensible ATP assays are available to get results.

2. The above suggested ibidi micro-slide technique is also great. In this case you can also evaluate the migration of cells when you can track the path of individual cells in a microscope by computer. For this you can use the free software Image J to evaluate the tracks.

3. As you have adhesive cells, you can look for somebody who has ECIS (Applied BioPhysics) equipment. In this one gold electrodes help to analyse adhesion of cells on the basics of impedimetry. When you have a confluent layer on the arrays, you can use two strategies (a) wound healing - in this case the equipment burns the layer above the spot of electrode and you can follow the migration of cells onto the cell-free region, (b) there is a "electric fence" function also, this keeps empty the electrode surface in the first part of the experiment - while there is a confluent cell layer everywhere but the electrode, then you turn off the fence and cells will start to move in. These are real-time assays and you get nice results about the migratory activity of your cells if you treat them compared to a control. (In this system the response is not chemotaxis but chemokinesis as there is no gradient of the tested chemical.

If you need any more info please visit our homepage www.chemotaxis.usn.hu.

Good luck!

Regards from,

Laszlo Kohidai

Dear Kelly-Ann,

Just out of curiosity, what kind of chemo-attractant are you using for your cells?

Try to pre-coat your transwell with "collagen. fibronectin....etc" .it depends on your cell type and their affinity towards the coating material.

What kind of cell do you use? what is the cell destiny? did you re suspend the cells in serum free media? what kind of chemo attractant did you use?