chemotaxis questions

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victorius
victorius's picture
chemotaxis questions

 Hi,

I'm doing chemotaxis assays with a microwell Boyden chamber and am having difficulty getting uniform cell migration, assessed by staining the polycarbonate filter and counting migrated cells: these are either at the peryphery of the well, forming a ring, or dispersed and packed in clusters, or even absent -although replicates nearby may have many cells. There are no apparent bubbles either below or above the filters, so: is there any rational explanation for this effect? Or is just my horrible luck? It seems also that the spots on the edges of the filter are the less reliable ones.

Before starting I heat the buffer/chemoattractants and vortex them. When filling the wells I'm taking good care not to get any bubbles,  but I think that -although hard to see -bubbles are introduced on top, when plating the cells. What can be done to minimize bubble formation? If I load 28 ul in the wells there are little or no bubbles but the medium spreads and reaches the contiguous  wells. Any practical advice will be appreciated. 

After migration I aspirate the cells on top, unscrew the chamber, invert the plate and clamp the filter, scraping the top side 3x as instructed by Neuroprobe. Then I fix in methanol and stain. I count several fields at 4x to 20x magnification, but not having uniform cell distribution is a good source of variation - and possible bias.  

Accesory questions: When using a receptor blocking antibody to pretreat the cells, is it best to incubate at 37C or at 4C? Should I wash out the antibody before plating the cells? 

I've read that PVP-coated filters are best for monocytes, but just a few labs seem to use these filters in those cells; any advantage of PVP-free filters over PVP-coated ones?

Thanks so much. 

heehawmcduff
heehawmcduff's picture
Hi there,

Hi there,

I'm sorry to hear you are having problems and I can certainly sympathize with you.  Chemotaxis assays can be incredibly variable.

I had a few questions and suggestions for you

1. Bubbles - depending on your chamber set up, it does require a very precise amount of liquid and it sounds like you have that figured out at 28ul.  A shortcut to getting rid of bubbles is to fire up a bunsen burner, open the valve to get a blue flame and wave it very quickly over the chamber.  However this is a poor substitute for good loading of the chemoattractant.

2. How long do you leave the cells to migrate?  This can greatly affect the results as leaving it too short gives you poor counts and leaving it too long can result in the cells migrating back through the filter.  Also are you using primary cells or cell lines?

3. I would recommend using transwells.  I know it is a lot more time consuming and you can test less samples than using a multiwell chamber but I found it to be far far easier to measure migration this way.

4. Pre-treat the cells at 37oC with your receptor blocker and you should definitely wash the cells before plating.

Best wishes

victorius
victorius's picture
Thanks so much heehawmcduff.

Thanks so much heehawmcduff. I really appreciate your advice.

I'm using a monocyte cell line. I used human monocytes too and migrated cells formed grape-like clusters (any idea why?).

All the Best.