LSRII #2: Your new best Friend, the Y-G Laser

One of the key attributes we wanted in our new LSRII was an excitation source in yellow-green (Y-G) spectrum. In this regard, there are basically 2 options. The first option is the 532nm Diode-Pumped Solid State (DPSS) laser, which has been in use for many years in flow cytometry, and the newly "usable" 561nm DPSS laser, which has gained popularity in the past 2 or so years. The benefits of any green/yellow laser is the ability to excite fluorochromes not excited by the standard 488nm or 633nm lasers commonly found on flow cytometers. There has been this huge gap between blue and red that has been neglected on mainstream analyzers for years. On sorters, people have used gigantic gas or dye lasers to get lines like the 532nm or 594nm which was ok for sorters, but those lasers don't really fit in the footprint of the bench-top analyzer. These green/yellow lines are needed for excitation of fluorochromes like Texas Red, mCherry (and the rest of the fluorescent proteins), and even Phycoerythrin (PE). What? PE? Yep, you read correctly. You may be thinking, but PE is my brightest fluorochrome, surely it is excited well by the 488nm laser. That may be somewhat true, but if you look at the actual absorption spectrum (http://www.bdbiosciences.com/spectra/) of R-PE, you'll see that at 488nm, R-PE is excited at only about 50% of its max absorption. At 532nm, it increases to about 80% of max, and at 561nm it's nearly 100% of max absorption. So, as you can see, the green/yellow lasers excite PE up to 2x better than a 488nm laser. What this means, is that not only will you be able to see fluorochromes like Texas Red and mCherry, but all your PE and PE-tandems will appear brighter off the 561nm laser than they would on a comparably powered 488nm laser.

And, if that's not enough to make you jump for joy, listen to this. Now that we're exciting PE off of the YG laser, and FITC off the blue laser, we have a built in temporal and spatial separation between the FITC emission and the PE emission. What does this mean??? NO COMPENSATION BETWEEN FITC AND PE!!!! That's right, the spillover of FITC emission into the PE channel is close to 0%. We all know that spillover reducing resolution of dim populations, so pairing better excitation of PE with no spillover from FITC means super resolution of dim PE stained populations. This is where the power of YG comes into play!

So, if you haven't figured it out yet, we went with the 561nm laser instead of the 532 laser. Here's a couple of reasons. 1. the 532nm laser interferes (to some degree) with the emission of FITC, so you'll need to use a notch filter to make sure you exclude the green 532nm laser light from the green FITC emission. 2. With the longer yellow-green emission, we can better excite PE, Texas Red, and a whole host of fluorochromes people want to use. 3. Laser powers are increasing for the 561nm. It use to be that you got the 532nm because the 561nm only gave you 20mW of power or so, whereas you could get 500mW of 532nm. There are now 75mW 561nm lasers available, bringing us closer to the coveted 100mW mark for lasers on flow cytometers, a point where we should be maxed out on excitation. 4. I actually tested the 532nm versus the 561nm on an LSRII at BD, and thought the excitation and "brightness" was pretty comparable even though the 532nm was at 200mW and the 561nm was at 50mW. Given comparable PE "brightness" we went with the longer wavelength laser in order to hit more fluorochromes our users are interested in.

Got more questions about this feature of the new LSRII #2, shoot us an email @ ucflow@gmail.com

LSRII number 2

In the coming weeks, the Flow Cytometry Facility will be getting its second BD LSRII. This instrument will have some additional lasers and fluorescence detectors compared to our current instrument. We're really excited about the potential of this instrument. The laser choices are what makes this instrument unique amongst all our analyzers. In the next few posts, I will be familiarizing you with the capabilities of this new instrument so you can prepare your experiments to take full advantage of what it has to offer. Some of the items put forth won't be unique to this instrument, but will go over things pertinent to the DiVa system in general. I'll try to point that out as each topic presents itself.

Need FlowJo Help?

The Flow Facility has expert knowledge in data analysis using FlowJo. You may have some specific questions on how to do something using the software, but you also may want help creating a workflow that is streamlined and efficient. This is exactly where a bit of knowledgeable help can really save you time. Many times people will continue to do repetitive tasks without realizing there is a way to automate things. For instance, even when doing a simple experiment a couple of times, it helps tremendously to use the template functionality inside FlowJo to help streamline the analysis process. Why not set up a time with the Flow Facility to allow us to help you streamline your analysis? We can do this kind of training one-on-one, or better yet, why not invite us to your lab meetings for a group presentation and troubleshooting session? If this sounds like something you'd be interested in taking advantage of, shoot us an email (ucflow@gmail.com), or give us a call at 702-9212. Fees may be charged for groups outside the University of Chicago Flow Facility User Base.
Don't have anything specific you need help on, but are interested in learning more about FlowJo? We'll give you or your lab a crash course on the basics of FlowJo. Otherwise, browse out our FlowCast archives for tips and tricks using FlowJo

The PCR Song...Hilarious

Since a lot of you folks use cell sorting for subsequent PCR analysis, I thought you might appreciate this song on YouTube. My Favorite parts are when the guy kisses and hugs the PCR machine, and the line "PCR, for when you need to know who the daddy is." Very witty, and very funny. Enjoy!

Flowcast? What's a Flowcast?

Flowcast is a derivation of the now popular term, screencast, which in and of itself is a derivation of the term broadcast. Now, although there really isn't any "broadcasting" of the screen in a screencast, the term simply refers to a tutorial or guide that uses a screen capturing program so you can see exactly what's going on and follow along easily. These screencasts are then broadcast on the internet for all to see. These types of tutorials work (for most) much better than simply reading through a protocol or document of directions. Flowcasts, therefore, are screencasts relating specifically to flow cytometry. To this avail, the flow cytometry facility has begun producing short (<10 minute) tutorials on whatever strikes us at the moment. We envision having mostly tutorials on how to do certain tasks in FlowJo and FACSDiVa, because that's what we mostly work with, and that's what most of the questions that are asked of us deal with. You'll see in the right hand quick links bar of our homepage (ucflow.uchicago.edu) a link for Flowcast. Check it out and let us know what you think. We would also like to encourage anyone who has an idea or trick that they like to use, let us know, and we'll make a flowcast out of it. The easiest way to contact us is at the following email address: ucflow@gmail.com

The significance of Significance

Did you ever wonder why we always collect 10,000 events in our files? Why not collect less? Or more? Well, to answer this question, you'll need to take a step back and stretch your brain to remember that pesky stat class you took as an undergraduate. You may remember that French mathematician with the funny name, Poisson. Well Poisson's distribution described the probability of a number of events occuring in a fixed period of time IF the events occur with a known average rate AND are independent of the time since the last event (don't believe me, check for yourself on Wikipedia). With that in mind, we'll need to assume that the events going through the flow cytometer pass through the instrument at a given and stable rate in order to allow us to apply the Poisson distribution to our flow data. Not a very difficult assumption knowing that our sample is pretty evenly distributed in a fluid, and that fluid passes through the instrument at a given flow rate. So, applying Poisson, the random and discrete occurrences or "arrivals" are the cells passing by the laser intercept, one by one. Also, Poisson tells us that if N events, or "cells", are observed or "collected" then the standard deviation (SD) associated with the collection is the square root of N. Brilliant! Additionally, we can express the SD in a more friendly term, the coefficient of variance (CV), which is 100 x SD/N. The CV is will give us a percent variance of our distribution. Very tight distributions will have a low CV, and very broad distributions will have a large CV.

Now, how is all this pertinent to flow cytometry. Well, lets say you collect your 10,000 cells, and you are analyzing a subset within that 10,000 cells that is at a frequency of 10%. This means, you've collected 1000 cells in your subset. The variance you want to measure and test the significance of is the 1000 cells in your subset, so, Poisson tells us that the SD of this population is equal to the square root of 1000, which is 31.62. If the SD is 31.62, then the CV is 100 x SD/1000 or 3.16%. This means that the accuracy of the frequency you report on the 1000 cells you collect has a variance of 3.16%. If you would like your CV limited down to say, 1% variance, then you would need to collect a data file of 100,000 cells, giving you a subset of 10,000. Given this simple equation, you can calculate ahead of time just how many cells you need to collect in order to achieve a certain amount of variance. A nice table and description is available at the following link from the folks at Cardiff University in the UK. So, in answer to our 1st question, we need to collect as many cells as will allow us to derive conclusions from our data that is backed up by significance.

RoboSep Demo Unit Available in Flow Lab

If you're doing any magnetic separations using beads from StemCell Technologies, or Miltenyi, or even Dynal beads, this unit may be of interest to you. The RoboSep from StemCell Technologies uses a very simple design to automate the pipetting necessary to couple your cells of interest to the paramagnetic beads. Once coupled, the built-in magnet will fractionate the coupled cells and the uncoupled cells allowing you to positively or negatively select your cells of interest. There are 4 magnets in the unit allowing you to fractionate up to 4 samples simultaneously. The touch-screen interface allows easy setup of samples and incubation times. The utility of the "magnetic sorters" can be maximized in the case that you wish to sort cells in the facility. A pre-enrichment of the population of interest can be done magnetically allowing you to decrease the amount of time on the sorters.

During the time that the facility has the demo unit, we will ask anyone interested to please try it out. Our StemCell contact is Rory Connelly (rory@stemcell.com). He has agreed to assist you with getting your samples run on the instrument, and the flow facility will allow you to re-run your samples on the analyzers free-of-charge (to check purity and yield post sort). We would also like to get feedback on the usefullness of such an instrument in the core. Would you realistically travel to the core facility to do your magnetic separations? Does it actually work for your project? This information will be very useful as we evaluate the instrument.

Feel free to contact us in the flow lab as well if you want more information