It's Here!!! LSRII-Blue

Well, the LSRII was delivered yesterday (3/25/08) and should be installed sometime this week. Once we get everything set up, we'll make it available for use. To differentiate between the 2 LSRIIs, we've decided to call them by their characteristic stripes, Blue and Orange. Our current LSRII is actually THE prototype LSRII created in-house at BD by Larry Ducket back in ~1999. The LSRI had an orange stripe, and the prototype was simply gutted and rebuilt inside to an LSRII. So, our LSRII is one of the only LSRIIs that has an orange stripe on it. It is therefore fitting to call our current LSRII, LSRII-Orange (or simply Orange), and our new LSRII, LSRII-Blue (or simply Blue). So, if you have a question about our LSRIIs just make sure you specify Orange or Blue!

LSRII #2: Best Color Combos

Considering this LSRII will have some of the same lasers/filters as our other instruments, you may be thinking the color combinations will probably be the same. Well, you'd be right, but maybe not for the right reasons. Here, I'll explain the best color combos, and why.

Just for redundancy's sake, let's take a look at what's available. A 405nm with 3 PMTs, a 488nm with 2PMTs, a 561nm with 4 PMTs, and a 640nm with 3PMTs (4 lasers, 12 colors). So, let's say you want to do a 12 color experiment, which colors will you use.

Let's start with the blue. Off the 488nm laser you're options are going to be FITC, PerCP or PerCPCy5.5. Now, if you're going to use PECy5.5 off the YG laser, then you'll want to use PerCP off the Blue instead of PerCPCy5.5. If however, you will use PECy5 off the YG, then you'll want to use PerCPCy5.5 off the blue. Next, we'll tackle the red. Off the Red laser, your options are APC, APCCy5.5 or Alexa 700, and APCCy7 or APCAlexa750. We have a similar situation as before, you'll want to repeat "Cy5.5" as few times as possible. Additionally, you'll want to avoid repeating the same emission spectra off different lasers as much as possible. Cy5 and APC have the same emission, so you'd want to avoid using PECy5 and APC together. APCCy7 is not that great, so you probably want to opt for the APCAlexa750 option. Now, for the yellow-green (YG) line. Any of the PE and PETandems would be appropriate, so you'll have PE, PETexasRed, PECy5, PECy5.5, PECy7, PEAlexa610, etc... Again, pick emissions that you have not duplicated elsewhere. Finally, you have the violet. For the violet your choices are Pacific Blue, Pacific Orange, Qdots, or dyes like DAPI. Special care should be taken when choosing Qdots as most of the Qdots will be excited by the blue laser and maybe even the YG laser. They also have high quantum yields, so even if they get excited by a non-optimal laser line, they'll still be pretty bright. You should again try to use the Qdots in places where you have gaps in the emission of your other fluorochromes.

So, with all that said, let's pick a panel. I'm going to propose PacBlue, Qdot 565, Qdot 625, FITC, PerCP, PE, PEAlexa610, PECy5.5, PECy7, APC, Alexa700, APCAlexa750 for my 12 color assay. I chose the two Qdots because they fill the gap between FITC and PerCP, and are less likely to be excited by the YG or Red laser. This 12 color combination will offer the greatest sensitivity with the least amount of compensation requirements. If however, you need to look at fewer colors, you could envision a panel of maybe 6 or 7 colors requiring little or no compensation. Here's an example: Pacific Blue, Qdot 625, FITC, PerCP, PE, PECy5.5, APC. This 7 color panel would have little to no compensation necessary whatsoever. Pretty cool, eh?

LSRII #2: What can I do with a Violet Laser?

The violet laser (typically a 405nm solid state) has become pretty much a standard laser on today's flow cytometers. The violet is typically cheaper and possibly longer lasting than a true UV laser. However, you may be asking yourself, what can I do with this laser line? The answer, Lots! Some fluorochromes have been specifically designed around the 405nm laser line, others, just happen to work well enough with it. Some common ones in the former group include Alexa 405, Pacific Blue, Pacific Orange, and Violet DyeCycle, while those in the latter group include DAPI, and Quantum Dots. For many years, people used UV sources on their flow cytometers simply to do "specialty" assays like Hoechst efflux (Side Population) or Calcium Flux (Indo-1) or just plain old cell cycle analysis (DAPI). However, no one really used the UV for immunophenotyping since the UV-excitable fluorchromes coupled to antibodies weren't bright enough. Now, with the necessity for doing more and more colors, we've run out of room on our Blue, Green, and Red lasers so we need to start using the lower wavelength lasers for more than these few specialty assays. The 405nm laser therefore allows us to open up the possibilities of more and more colors. Simultaneously, we could conceivable look at Pac Blue, Pac Orange, and a Q-dot 705 conjugate. This gives us 3 more usable channels for our multicolor experiments. Or, once Q-dots becomes readily available in direct conjugates, then you could use a few Q-dots in these channels. Also, the we've found the violet laser to work just fine for DAPI, even for cell cycle analysis. You don't get as good of CV's as you might with a true UV, but it's pretty decent (G1 CV<5.0).

So, what do you lose with a UV? Side Population with HO 33342 is not good at all. Maybe it's ok on bone marrow, but that's about it. But, there are alternatives. You could do side population with the violet dyecycle dyes from Invitrogen, or you could use the other LSRII with the UV laser on it. The other thing you lose is Indo-1. There's no way you're gonna be able to do any indo-1 on a violet laser. But, you can use other calcium sensitive dyes like Fura-Red and Fluo-3. These are blue excitable, and when used together, you can get similar ratiometric measurements as you would with indo-1. If you're a BFP user, switch to CFP or cerulean.

Other than the few things mentioned above, the absence of a UV laser may not be that bad depending on what type of user you are. Please note however, we will have a UV on our other LSRII for at least the near future, so if you need to use UV, you're still in luck!

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!