Hi, we have a protocol from a manufacturer of a Ca2+ flux assay which requires macrophages be incubated with the dye (FURA-3) in sterile PBS without Ca2+ or Mg2+ for 1 hour at 37C. We noticed that often our fully confluent macrohages round up and detach after this hour. This time we incubated the macrophages in sterile PBS-- on its own for the hour and the cells still detached, showing it is the PBS (commercially bought) and not the dye causing the cells to round up.
Is there a reason for this and can anyone suggest an alternative diluent?
We are going to try PBS we make ourselves from tablets and sterilise.
Thanks
Hefin
it could be the same reasons why we use EDTA-trypsin (not trypsin alone) for detaching cells during passaging. Calcium dependent cell-cell adhesion is known.
http://jcb.rupress.org/content/105/6/2501.full.pdf
Maybe you can try slightly more complex buffer like HBSS (Hank's balanced salt solution, good for mouse macrophages I worked with) or Tyrode buffer (good for cardiomyocytes).
The Tyrode recipe is simple, and you can withhold Ca2+ and Mg2+ during preparation: http://cshprotocols.cshlp.org/content/2007/2/pdb.rec10805.full?text_only=true
There is a commercial HBSS from Invitrogen that is calcium and magnesium free.
https://www.lifetechnologies.com/order/catalog/product/14170112
Another way is to pre-coat your coverslips with PEI, followed by UV sterilization.
PEI forcibly retains cells on a coated surface (some cells exhibit PEI toxicity however). So find that out empirically.
I'm totally agree with Nai-Kei Wong - much better to use HBSS (ion composition and glucose prevent cell degradation).
Additionally test yours plastic (suspension culture plastic should be excluded). From my experience better to use "cell+" or treated for adhesive cultures to prevent cell rounding and detaching.
It also could help if you coat your plastic with collagen before experiment.
i agree with Nai-Kei Wong. It is better to use Ca2+, Mg2+ free HBSS. Further you can try using dye Fura 3AM/ Fura 4AM for Ca2+ flux assays. It has much shorter time for loading. If you determine flux by FACS, you can use both together as they are diametrically opposite dyes.
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