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!