Re: [DIYbio] Got FACS expertise?

[Nathan McCorkle]

I'm no expert but I've sat in front of a newer working FACS before. I remember hearing a big part of running them is the sheath buffer, PBS I think it was, gets used once then goes to waste. The lasers for fluorescence can get dim and/or die out altogether. There are likely high-voltage PMTs inside, probably three for each laser color (it may only be a single color machine, but google results show it might come standard with more)... you get back-scatter forward-scattter and side-scatter data, these data channels can be placed on an XY cartesian plane and graphed with false-color representing number of events. So if you get a lot of particles/cells fluorescing the same in side and forward-scatter, there will be a bright spot on the XY plot, and you can draw a conclusion that it is a single type of particle responsible. If you had mixed 2 cell types and you wanted to separate them, you would need to play with your assay (i.e. what dyes you're using) so you'd see two bright spots on the XY plot. Then you basically just draw a circle around the cell group you want to keep, and any events that fall within your circle will get collected, the rest will go to waste.

If it has the FACSworkstation software for running the machine, you could be up and running in a few days. You could probably use distilled water instead of PBS if you're just using calibration plastic microspheres (bangslabs.com).

http://www.cytometryres.com/news/print/02q2-p3.pdf

http://crl.berkeley.edu/flow_cytometry_basic.html

Worst case is you get a bunch of cool stuff to hack on! I see at least one or two tektronix oscilloscopes!

On Thu, Nov 15, 2012 at 6:41 AM, Emil Polny <emil...@gmail.com> wrote:

Hi all, 

In BiologiGaragen/Labitat in Copenhagen we have been offered a Fluorescence Activated Cell Sorting (or FACS) machine as a donation to the DIYbio space (see the photos below). 

This is a very generous offer, but none of us have the necessary experience to set it up, calibrate it, etc. 

Do anyone of you have this kind of experience? 

Could you share it with us? 

Thanks a lot in advance, 

Emil

biologigaragen.org

labitat.dk

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-Nathan

[plushzilla]

Hi Emil,

It's a very generous donation! They are actually pretty expensive to run and maintain, and it brings back good old memories of cell sorting experiments from my postgraduate days.

As Nathan mentioned before, a big part of the experiment is running the right type of control and working out the right set of fluorescent markers to use so that your results can be interpreted correctly. What type of application would you be thinking about using it for? I have had some experience with both the standard markers as well as the bangs beads, and generally they are not too difficult to use. The suppliers normally are also very helpful, because they want to build a good relationship to keep the orders for consumables ticking along.

If you have any more information I'd be happy to provide some suggestions and advice.

Thanks

[Jonathan Street]

A generous donation indeed. Do you know what condition it is in? I believe the state-of-the-art is marching along swiftly so hopefully this is from an earlier generation but functional (or nearly so).

I've done quite a bit of work with flow cytometry but never cell sorting. I suspect BD would be willing to give you quite a bit of help in the hope of consumables sales later down the road.

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