University of Chicago Cancer Research Center achieves Comprehensive Status

For those of you who are not aware of the University of Chicago Cancer Research Center (UCCRC) on campus, I'll share a bit of good news. First of all, the Cancer Research Center Support Grant has been renewed for 5 years, and secondly, the UCCRC has achieved Comprehensive status. This is sort of a big deal, considering in the entire country, there are only 41 Comprehensive Cancer Research Centers, and in Illinois, there are only 2 (the other one being the purple people downtown). Being a Comprehensive Cancer Research Center basically means we've got really great researchers and support staff working on attacking malignant diseases, or as the NCI likes to say, "Comprehensive Cancer Research Centers are characterized by scientific excellence and the capability to integrate a diversity of research approaches to focus on the problem of cancer."

So, what does all this have to do with flow cytometry? Well, a big part of the UCCRC grant was the availability and access UCCRC members have in terms cutting-edge technology and technical expertise. A big chunk of the funding goes towards operational costs of core facilities and subsidized usage fees for member investigators. The flow cytometry facility, having a large number of cancer research investigators has been the recipient of such funding for over a decade. Over 90% of the usage of the facility is performed by members of the UCCRC, so we are very much an integral part of the UCCRC and its newly achieved Comprehensive status. More info on the UCCRC can be found online at http://uccrc.uchicago.edu or if you'd like more info on the NCI Cancer Research Center Program, you can visit http://cancercenters.cancer.gov. Congratulations to all involved!

ISAC Wrap-up Part 2

Along the same vein as the previous post, the Education committee presented an outline of their plans to bring flow cytometry education to the general scientific public. In an attempt to standardize the information being presented to users of flow cytometry, the Education committee has decided to generate a basic flow cytometry course aimed at flow novices. The course would initially be offered as a tutorial tacked on to the front end of other disciplines' meetings, who have used flow cytometry in the past. From there, plans to make it available as an online course administered through ISAC were discussed. Lastly, it was proposed that this course become the template for training used by core facility directors in training their user base. The Education committee still seems to be developing these ideas and reworking their strategies, so it may be awhile yet until we see anything concrete. Don't know yet if there will be an official "accreditation" process for this type of course, but if and when it's possible, we'll be sure to take a good look at the material to see how we can integrate it into our educational process.

ISAC Wrap-up Part 1

Planning to submit a paper to J. Exp. Med. anytime soon? Well then you'll want to pay attention to the new guidelines generated by the Data Presentation Standards Committee and adopted by J. Exp. Med. What the cytometry field has been noticing for quite a while is the amount of publications in high-end journals with really poor flow cytometry figures. It's not that the data is bad, or doesn't support the authors conclusions, it's just that the figure is annotated so poorly, and the methods written so vaguely that it's nearly impossible for anyone to try and repeat complicated analyses. Being the premier flow cytometry authority, ISAC has taken it upon itself to lay down some guidelines to assist authors in properly presenting the necessary information to describe their flow cytometric data. A member of the Data Standards Committee, Dr. Mario Roederer, shared the conclusions of the committee this week at the ISAC Congress. Here are a few examples from the guidelines. Once a comprehensive list is created, I'll post them for the group to see.

Author must list the instrument(s) used, the laser(s) used for excitation, and the filter(s) used for emission. This will be done in an excel template that will be available from ISAC or J. Exp. Med.

Axis on plots must have the reagent and fluorescence labelled (ex. CD3-FITC).

The number of events in a plot must be displayed in the plot or figure legend.

The frequency of populations must be displayed on the plot or in a table

Graph-types should be used consistently throughout.

You MUST show the entire gating tree for your figure as an example. Back-gating analysis is the best way to show this. This can (and probably should) be in the supplemental data portion of the manuscript.

You must also state how you drew your gates. For example, did you draw them based on unstained cells, or an isotype control, or and FMO control, or did you just subjectively draw it around a population.

These are a few of the guidelines that will be implemented soon. J. Exp. Med. has decided to adopt them sooner rather than later, but we anticipate more and more journals will begin to require this information. If you have any questions, or need help filling in the appropriate information, the flow can certainly help. We will try to provide filled out examples for each of the instruments so that you can simply modify them for your use. We'll provide more info as it becomes available.

Greetings from Budapest

This year's ISAC meeting, held in Budapest, Hungary is well under way. I'll post some interesting tidbits of info as the meeting progresses. One interesting tidbit is that ISAC has changed it's name effective immediately. It will still retain the acronym ISAC, but the last two letters have been slightly modified. Previously, we were known as the International Society for Analytical Cytology. That name, analytical cytology, doesn't really fit with what the society does, so it was voted on and it was decided the new name will be "International Society for Advancement of Cytometry" Cytometry is what we do, that is, measure cells. Also, our goal is to advance the field of cytometry, so the name makes much more sense. More later.

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!