MIT Flow Cytometer Core Facility

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[http://www.cyto.purdue.edu/flowcyt/labinfo/images/TutorialWinMDI.pdf WinMDI tutorial]
[http://www.cyto.purdue.edu/flowcyt/labinfo/images/TutorialWinMDI.pdf WinMDI tutorial]
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[http://www.burle.com/cgi-bin/byteserver.pl/pdf/Photo.pdf Photomultiplier Handbook] - all you ever wanted to know about PMTs

Revision as of 21:05, 20 March 2006


This information is largely gleaned from the Flow Facility Homepage. As well as conversations with Glenn Paradis.


Contents

Flow Cytometers

FACScan

FL1: 530/30

FL2: 585/42 (this means 585 +/- 21)

FL3: 650 longpass

Laser: 488nm

Using 2 Fluorescent Proteins in the FACScan cytometers

As of now it seems the best choice is GFP/dsRED, however you will be exciting dsRED off the 488 line (30%). So you need bright cells. It is unlikely that GFP/mRFP1 will work since the mRFP1 spectrum is shifted (5% excitation at 488), though this is currently untested. CFP/YFP will not work since there is not a line that effectively excites CFP.

High Speed Sorters

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, however with some adjustment they can be made to sort bacterial cells.

Using 2 Fluorescent Proteins in the FACS machines

It seems that the only FP pair that will work with the current setup in the flow facility is dsRED/GFP (or mRFP1/GFP, although this has not been confirmed). CFP/YFP will not work because they are getting poor excitation of CFP on the FACS-Aria. It seems like this is a problem on their end since I can see strong expression on the scope. The MOFLO has a (?) line which is appropriate for RFP expression and the 488 for GFP.

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.

Question How do you convert the signals recorded by the FASC machine that were logarithmically amplified electronically before digitization, and are now represented on a linear "channel numbers" scale into something more appropriate for data analysis?

Answer http://www.cyto.purdue.edu/hmarchiv/1999/1521.htm

Other Resources

Software

Spectra Viewer

Statistics

WinMDI tutorial

Photomultiplier Handbook - all you ever wanted to know about PMTs

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