I've decided to post to the New England Cytometry blog instead of here, so if what I've written about flow is helpful to you, I hope you'll follow me there!
www.newenglandcytometry.com
Just copying over some of the topics I've covered here so far, with new material on the horizon...
Mike
Monday, December 31, 2012
Monday, August 29, 2011
Compensation
Due to popular demand (ok, one or two people mentioned it), I'm going to try to add more content to this blog and maybe help teach people about flow cytometry. There has already been another ISAC Congress held, so I've given up on posting about ISAC 2010, maybe next time I'll do it DURING the meeting. I'd also been composing a post and saving it to finish the whole thing, but perhaps I should post in smaller chunks to actually get something published.
I think compensation makes a good topic for my first blog post for flow topics, as it can have the greatest impact on your data and is somewhat tricky if you dont understand the process. There are lots of good online tutorials and posts about compensation linked from our "Training" page from our website (www2.massgeneral.org/aids/flow_cytometry.html) but there are a few specific items I'd like to address and have "published" for viewing so I'll throw my hat in the ring as well.
All fluorochromes will emit light across a range of wavelenghts, and if you are trying to take measurements for another fluor in a band that is included in this range, you will get background light ("spillover" or "spectral overlap") from that other fluorochrome. To correct for this, most cytometry software has some mechanism for "compensation" to remove this background. In essence, for any other parameter/detector
that you are measuring, you want your single positive population to have the same Mean Fluorescence Intensity (MFI) as the negative population so that any positive signal in the other channels can be attributed to that particular color, and is not spillover from something else. In the case shown, FITC signal is being detected in the PE channel, and once compensated has the same mean fluorescence in the PE channel as the unstained.
Many companies now offer antibody-capture bead kits for compensation (Invitrogen, BD, Bangs Labs, Spherotech) and they usually include a "binding bead" and "non binding bead" in the same box. For example, BD offers "Compbeads" which bind to a specific species' IgG (mouse, rat, hamster), so for a mouse-anti-Human CD4 antibody, you'd need the mouse binding bead, but for a rat-anti-mouse CD19 you'd need the rat binding bead. For Invitrogen's ARC beads which are used to compensate their amine-reactive Live/Dead fixable stains, the binding beads are coated with amine groups so the reactive dye will bind to the bead. The Live/Dead dye will NOT bind to antibody-capture beads.
In order to correctly do this calculation, it is critical that you are comparing particles of the same type-- if you are using stained cells, you want to compare the signals to the *same type* of unstained cells. If you are using beads, you want to compare to the exact same unstained bead. The "non-binding" bead is included in the kits for this purpose-they are the same bead as the binding one in the kit, but will not stain with the reagent and thus remain negative. You can include them both in the same tube, although you should wash the beads after staining to remove free dye if you do so.
Mismatches in your positive and negative particles will result in compensation errors, which could dramatically impact your data. More on that next post...
I think compensation makes a good topic for my first blog post for flow topics, as it can have the greatest impact on your data and is somewhat tricky if you dont understand the process. There are lots of good online tutorials and posts about compensation linked from our "Training" page from our website (www2.massgeneral.org/aids/flow_cytometry.html) but there are a few specific items I'd like to address and have "published" for viewing so I'll throw my hat in the ring as well.
All fluorochromes will emit light across a range of wavelenghts, and if you are trying to take measurements for another fluor in a band that is included in this range, you will get background light ("spillover" or "spectral overlap") from that other fluorochrome. To correct for this, most cytometry software has some mechanism for "compensation" to remove this background. In essence, for any other parameter/detector
that you are measuring, you want your single positive population to have the same Mean Fluorescence Intensity (MFI) as the negative population so that any positive signal in the other channels can be attributed to that particular color, and is not spillover from something else. In the case shown, FITC signal is being detected in the PE channel, and once compensated has the same mean fluorescence in the PE channel as the unstained.
Many companies now offer antibody-capture bead kits for compensation (Invitrogen, BD, Bangs Labs, Spherotech) and they usually include a "binding bead" and "non binding bead" in the same box. For example, BD offers "Compbeads" which bind to a specific species' IgG (mouse, rat, hamster), so for a mouse-anti-Human CD4 antibody, you'd need the mouse binding bead, but for a rat-anti-mouse CD19 you'd need the rat binding bead. For Invitrogen's ARC beads which are used to compensate their amine-reactive Live/Dead fixable stains, the binding beads are coated with amine groups so the reactive dye will bind to the bead. The Live/Dead dye will NOT bind to antibody-capture beads.
In order to correctly do this calculation, it is critical that you are comparing particles of the same type-- if you are using stained cells, you want to compare the signals to the *same type* of unstained cells. If you are using beads, you want to compare to the exact same unstained bead. The "non-binding" bead is included in the kits for this purpose-they are the same bead as the binding one in the kit, but will not stain with the reagent and thus remain negative. You can include them both in the same tube, although you should wash the beads after staining to remove free dye if you do so.
Mismatches in your positive and negative particles will result in compensation errors, which could dramatically impact your data. More on that next post...
Tuesday, July 20, 2010
ISAC
Back in May I attended the ISAC Congress in Seattle and had the (admittedly unoriginal) idea to do a daily blog about what I saw/heard while I was there, but forgot the power cord for my laptop and so by day 2 had no battery power left... It was going to be LEGEN....wait for it.... ah never mind, here it is 3 months later and I still haven't posted anything about it, but I'm carrying around a scrap of paper with some notes on it of stuff I wanted to post so I'm going to try to put some sort of writeup here over the next few weeks. Hopefully someone will find it helpful and/or amusing.
So it was the XXVth (is that even a term? Zzzvith? Chchivith in Nahuatl) ISAC Congress, and just in time since my computer bag from the last North American congress (Quebec City in ’06, they alternate every 2 years between N.A. and Europe and I was unable to go to Budapest in ‘08) was just about worn out. I don’t know what they were thinking this year, there are 5 weekends in May and they have to pick Mothers Day weekend? But anyway, the website for the event is http://www.cytoconference.org/ if you want to see the lineup, and, eventually, the presentations themselves I hope.
The first thing about Seattle is the number of coffee shops. There is one on every block. The second thing is the number of disposal sorting options you have, at least at the convention center, which makes throwing away your coffee cup that much more difficult. Each trash bin was divided into Bottles/trash/compostables-- so is the plastic lid recyclable in the bottles bin? can you compost the paper cup or is it trash?
I also don’t get the rationale of running the meeting from Saturday to Wednesday? Have to take Friday off to fly out, and get back on a Thursday? I guess I can’t really complain unless I’m willing to sign up for the planning committee, so we’ll just let it go. I will complain about the bags this year tho, they are pretty flimsy and the plastic clip for the shoulder strap unclipped a few times, dropping the bag to the floor (thankfully my laptop was NOT in it at the time, since it had no power!).
I signed up for a few of the pre-congress sessions. First up was data analysis, which as advertised was very basic, but since I hadn't taken a "formal" data analysis seminar before, was still useful. The most interesting bit I got out of it was that you are really only detecting *2%* of your starting light on a typical cytometer-- collection lens gets about 15%, about 60% of that gets through the filters and mirrors, and then the PMT efficiency is around 20%, so a lot of signal is lost.
The other bit I'll talk about here is another artifact of log display called the "Valley Artifact". Anyone using a digital instrument should know about the "Picket Fence" where the first decade of data ends up with lines of data since digital data will only have whole-number values and you only have 10 values represented in the first part of the display.
The "Valley Artifact" was not seen in earlier systems because log amps were quasi-linear at the low end. But in digital, there is no log amp since log/lin is just a display mode, and as the signal is decreased, the peak will get "stuck" at a value and just shrink in size while more events pile up in the first channel. You have too much resolution in the low end with not enough events to fill it. There were a lot of other topics covered but if you are interested you can take a look at my notes, or wait for the presentation to be posted.
The next talk I had seen a few times before, Instrument and ICS Standardization for Polychromatic Flow by Steve Perfetto and Pratip Chattopadhyay, from the VRC at the NIH. They also host a weeklong workshop dealing with these topics and issues, called VALID-PFC that I was able to attend a few years ago. There are so many different components that matching instruments is very challenging, and how to best do that is still being worked out.
The next tutorial was Managing a Core Flow Facility, which will only be interesting to maybe 1% of cytometer users, and ran the gamut of topics on how to organize and run a core efficiently, how to fund such an endeavor, and other concerns in keeping users happy and your core in the black.
The State of the Art Lectures followed opening remarks, and started with Mario Roederer talking about Advanced Multiparameter Analysis, mainly the Cytof which saw a good amount of coverage and I'll get to later.
Chen-Yuan Dong spoke about In Vivo Fluorescence Microscopy and Spectroscopy, and an article in the Biomedical Optics Journal about inserting a window in a mouse's abdomen and imaging live organs/cells in the animal.
Krishan Dholakia's topic was Advanced photonics and light manipulation of cells, using laser beams to actually move cells around and even photoporation to introduce exogenous DNA into cells. http://www.st-andrews.ac.uk/~atomtrap/
The Celebration Lecture by Alexandra Worden discussed Understanding Algal Roles in Global CO2 Uptake, which made use of oceangoing flow cytometers. Next we need to get one on a plane, or perhaps outer space??
For dinner the contingent of cytometrists from Boston hit the Taphouse Brewery, conveniently located on the next street over from the hotel. They had 160(! ) beers on tap, and a sampler option of four 6 oz glasses. Another point in favor of the place was that their playlist included Guster!
Boston Band count: 1
So you'd need to get 40 samplers to make it through all of their tap beers, I opted for a regular glass the first night and tasted a few others. Beer count: 3.
So it was the XXVth (is that even a term? Zzzvith? Chchivith in Nahuatl) ISAC Congress, and just in time since my computer bag from the last North American congress (Quebec City in ’06, they alternate every 2 years between N.A. and Europe and I was unable to go to Budapest in ‘08) was just about worn out. I don’t know what they were thinking this year, there are 5 weekends in May and they have to pick Mothers Day weekend? But anyway, the website for the event is http://www.cytoconference.org/ if you want to see the lineup, and, eventually, the presentations themselves I hope.
The first thing about Seattle is the number of coffee shops. There is one on every block. The second thing is the number of disposal sorting options you have, at least at the convention center, which makes throwing away your coffee cup that much more difficult. Each trash bin was divided into Bottles/trash/compostables-- so is the plastic lid recyclable in the bottles bin? can you compost the paper cup or is it trash?
I also don’t get the rationale of running the meeting from Saturday to Wednesday? Have to take Friday off to fly out, and get back on a Thursday? I guess I can’t really complain unless I’m willing to sign up for the planning committee, so we’ll just let it go. I will complain about the bags this year tho, they are pretty flimsy and the plastic clip for the shoulder strap unclipped a few times, dropping the bag to the floor (thankfully my laptop was NOT in it at the time, since it had no power!).
I signed up for a few of the pre-congress sessions. First up was data analysis, which as advertised was very basic, but since I hadn't taken a "formal" data analysis seminar before, was still useful. The most interesting bit I got out of it was that you are really only detecting *2%* of your starting light on a typical cytometer-- collection lens gets about 15%, about 60% of that gets through the filters and mirrors, and then the PMT efficiency is around 20%, so a lot of signal is lost.
The other bit I'll talk about here is another artifact of log display called the "Valley Artifact". Anyone using a digital instrument should know about the "Picket Fence" where the first decade of data ends up with lines of data since digital data will only have whole-number values and you only have 10 values represented in the first part of the display.
The "Valley Artifact" was not seen in earlier systems because log amps were quasi-linear at the low end. But in digital, there is no log amp since log/lin is just a display mode, and as the signal is decreased, the peak will get "stuck" at a value and just shrink in size while more events pile up in the first channel. You have too much resolution in the low end with not enough events to fill it. There were a lot of other topics covered but if you are interested you can take a look at my notes, or wait for the presentation to be posted.
The next talk I had seen a few times before, Instrument and ICS Standardization for Polychromatic Flow by Steve Perfetto and Pratip Chattopadhyay, from the VRC at the NIH. They also host a weeklong workshop dealing with these topics and issues, called VALID-PFC that I was able to attend a few years ago. There are so many different components that matching instruments is very challenging, and how to best do that is still being worked out.
The next tutorial was Managing a Core Flow Facility, which will only be interesting to maybe 1% of cytometer users, and ran the gamut of topics on how to organize and run a core efficiently, how to fund such an endeavor, and other concerns in keeping users happy and your core in the black.
The State of the Art Lectures followed opening remarks, and started with Mario Roederer talking about Advanced Multiparameter Analysis, mainly the Cytof which saw a good amount of coverage and I'll get to later.
Chen-Yuan Dong spoke about In Vivo Fluorescence Microscopy and Spectroscopy, and an article in the Biomedical Optics Journal about inserting a window in a mouse's abdomen and imaging live organs/cells in the animal.
Krishan Dholakia's topic was Advanced photonics and light manipulation of cells, using laser beams to actually move cells around and even photoporation to introduce exogenous DNA into cells. http://www.st-andrews.ac.uk/~atomtrap/
The Celebration Lecture by Alexandra Worden discussed Understanding Algal Roles in Global CO2 Uptake, which made use of oceangoing flow cytometers. Next we need to get one on a plane, or perhaps outer space??
For dinner the contingent of cytometrists from Boston hit the Taphouse Brewery, conveniently located on the next street over from the hotel. They had 160(! ) beers on tap, and a sampler option of four 6 oz glasses. Another point in favor of the place was that their playlist included Guster!
Boston Band count: 1
So you'd need to get 40 samplers to make it through all of their tap beers, I opted for a regular glass the first night and tasted a few others. Beer count: 3.
Thursday, April 29, 2010
Welcome!
Thanks for visiting the blog of the Flow Cytometry Core Facility of the Ragon Institute of MGH, MIT, and Harvard. I plan to post different topics relating to flow cytometry, including technology, common issues, how-to's, etc. Any suggestions for topics you'd like to see please email ragonfacs@gmail.com and I'll try to work it in. For more flow cytometry related links and info on the core offerings please visit www2.massgeneral.org/aids/flow_cytometry.html or go to http://www.ragoninstitute.org/ and click on the "Flow Cytometry" link on the right side.
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