MIT Flow Cytometer Core Facility

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Revision as of 01:05, 9 June 2005 by Reshma P. Shetty (Talk | contribs)
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This information is largely gleaned from the Flow Facility Homepage. As well as conversations with Glenn Paradis.


Contents

Flow Cytometers

FACS

There are 4 cell sorters in the facility:

Excitation Wavelengths

  1. 488,633
  2. 407,488,633
  3. ?
  4. ?

There FACS-Aria machines (2 of the 4) have a system for creating the droplet which allows for smaller cells (i.e. bacteria) to be resolved. The MOFLO machines are not as capable in this regard.

FAQ

Question

I had a question about PMT settings. I am using the FACScan left to measure fluorescence from YFP in E. coli cells. I have been changing my growth conditions and whatnot and I noticed that my negative control cells are "more off" than they were before. ie A lot of them are piling up in the first channel. So I tried taking fluorescence measurements again using a high PMT setting (I went from Fl1 at 595 to FL1 at 690). As expected fewer of the cells are piling up in the first channel but some still are. At this new setting, distribution of cells is fairly well centered in the first quadrant between 10^0 and 10^1. Is it worthwhile to turn up the PMT setting even more so that I decrease the number of cells in the first channel even more? My positive control lands primarily in the third quadrant between 10^2 and 10^3 so I could arguable turn up the PMT settings and still not have cells pile up in the last channel.

Glenn Paradis' answer

No need for you to pull all the cells off the bottom of the axis unless you want to ratio the mean of the pos. vs. the neg. If you just want % pos compared to a neg control then having some cells on the axis is no big deal unless of course there is a lot of cells off scale (i.e. more than 20%).

Question

Just to clarify, is there any reason not to increase the PMT setting even more assuming none of my cells accumulate in the last channel? In other words, the primary factor limiting your PMT setting is the danger of having cells accumulate in the last channel?

Glenn Paradis' answer

There is no reason to keep the PMT voltage low. Crank away at 999 volts.

Other Resources

spectra viewer

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