Tuesday, April 14, 2009

Welcome back Calcium Flux!

Is it just me, or does there seem to be a resurgence of calcium flux assays being done these days. Back in the day (don't laugh, I've been around for a relatively long time) calcium flux was pretty much standard procedure for immunologists and cell biologists alike. However, for various reasons, it fell out of favor over the past few years. Now, it seems like every few days, someone comes to talk to me about doing some calcium flux. You're probably well aware of Calcium's role in pretty much any cell activity, and the 'rapid' influx of the ubiquitous cation upon activation; so for those assays where you need to determine whether some stimulus actually causes the cells to increase activity or if that surface protein tail you modified causes a lack of downstream activity, this may be the perfect assay.

The way this assay works basically involves a fluorochrome which undergoes some sort of structural confirmation change upon binding Calcium, resulting in either an increase/decrease in fluorescence or a change in fluorescence absorption/emission. So, if you load that dye into a cell sample, you can track this change in fluorescence over time and quantify the rate of flux (how fast calcium rushes in), peak calcium flux (how much calcium rushed in), or duration of flux (how long the flux lasted). Some examples of these dyes include the perennial favorites, Indo-1, Fluo-4, and Fura Red, as well as some multitasking dyes like Oregon Green, and Calcein. Of course Molecular Probes (Invitrogen) has a calcium sensitive indicator dye in pretty much any color you can imagine. In addition to the favorites, they have a group of dyes cleverly called Calcium Green, Yellow, Orange, and Crimson. They all have slight variations in their chemistry, but all do pretty much the same thing. Whenever dealing with dyes and fluorochromes, there is no better place to look than the Molecular Probes Handbook, especially if you want way too much technical information than you could every imagine. Here's the link to the calcium indicator dye section if you want more info. Chapter 19, if you're a seasoned veteran of the handbook. The one major caveat that I need to bring up right here and now is that Indo-1 requires a UV light source, and not just any UV light source, it's gotta be really into the UV spectrum. We used to have a 325nm HeCd source, which was perfect for Indo-1. The only bad thing was the laser would only last about 6 months, and then the CVs were way too wide to be useable. We played that game for a couple of years, but have finally given up (almost) on UV altogether. We have violet sources, but no UV sources. But, do not fear, there are tons of options in every color for fitting in a Calcium indicator into your flow panel. And for any flow aficionados reading this post, yes we've tried the lightwave 355nm quasi-CW solid state laser. They are way too expensive, and only lasted about 18 months for us...we went through 2 of them. Our latest covetous thoughts are pointing us towards a 375nm diode. Not useful for Indo-1, but possibly good enough for DAPI cell cycle. As always, if you want to do calcium flux assays, but don't know how to get started, feel free to contact us day or night, but we'll probably only respond during the day.