"Lasers"

Do you like Star Wars? I do. I'm not one of those freaky, dress-up like Chewbacca fans, and talk like yoda, I do not, but what I really like about Star Wars are the lasers. It just seems like such a practical way to shoot things. No need to carry along a bunch of ammunition, all you need is your trusty multi-KiloWatt laser. Another benefit is that light travels, well, at the speed of light, much faster than a speeding bullet or a falling bomb. Lasers are used all over the place, most-notably for me, Flow Cytometry and Microscopy, but now it seems like the vision of Star Wars may be closer than ever. Darpa (The advanced research division of the Department of Defense) has been investigating weapons-grade lasers for some time, but with the use of some newer technologies, there may actually be a viable option. HELLADS, or High Energy Liquid Laser Area Defense System, a laser system developed by Textron, is getting some big government bucks to design and test a 150kW Laser Weapon System that could be carried on a Fighter Jet and be used to shoot down missiles. So, what's so special about the laser? It seems this laser system is using a series of thin ceramic slabs, bathed in rapidly circulating coolant to achieve a high power output while maintaining a non-destructive temperature load. These so-called slab lasers are attempting to tackle a long-standing problem with lasers, what to do with waste heat. If successfully employed, these types of technologies may trickle their way down to us lowly folks in the flow cytometry world, and I may actually be able to realize my goal of a centralized laser depot with a bunch of fiber-optic cables extending out of it. I've already got a name for it...Medussa! If you have a high enough laser source, you could split that beam a bunch of times, and using fiber optic cables, bring the light to any number of instruments using a "plug-n-play" connection scheme. Some day...

Flow Basics is Back! and slimmer than ever!

That's right folks, by popular demand, we're reintroducing a legend, Flow Basics. But, Wow Flow Basics, have you been working out? I mean, you're about half the size as the last time I saw you. You've gotta be down to, what, like 5MB or something? Not only is Flow Basics back, but it's leaner and meaner than ever, well, not really meaner, but definitely leaner. We've trimmed a bunch of the slides while still focussing on the most important aspects of basic flow cytometry theory and instrument usage. We will once again be making this a mandatory requirement for new flow cytometer trainees, but anyone who did not attend the original flow basics, will want to attend one of these training sessions. To make this process easier, we will be offering the course more frequently especially in the next coming months. Once the initial rush is over, we will over the course every other week as new users are being trained. We're also putting the course schedule on the facility's Online Instrument Scheduler, so now, you'll know exactly when the next class will be offered, as well as have an opportunity to sign up whenever is convenient for you. Although the Online Scheduler offers convenience and flexibility, the sign-up process has a few steps to it that may make it somewhat challenging the 1st time you attempt. So, we also made a little video demonstration on how to sign-up for training. We've embedded the video here for you to watch. Classes start 12/10/08, so check the online scheduler for more dates and to sign up.

561nm Laser Demo - MoFlo

We will be having a demo of a 200mW 561nm solid state laser on our MoFlo December 2nd and 3rd, 2008. Beckman Coulter will be in to install the laser as well as a detector block so we can assess the utility of such a laser for our sorting applications. The major benefit of this laser will be the ability to excite fluorochromes not normally excited at 488nm or 633nm, such as mCherry, RFP variants, and more. Other benefits of interest will be the stronger excitation of PE and PE-Tandems as well as the lack of spillover of blue excited fluorochromes like FITC and PerCPCy5.5 into PE channels. This will effectively increase the resolution of dimly stained populations, especially when identifying cells which are double positive for FITC and PE. We have a few scheduled demos of the laser with users of the facility, so if you'd like to see the laser in action, give us a call to find out what time we'll be running samples on the 2nd and 3rd. Or, just drop by on either day to take a look at the setup.

New - Flow Data Flickr Image Pool

New - Flow Data Flickr Image Pool
Originally uploaded by UCFlow

Why not stop over at the UCFlow Image Pool on Flickr (www.flickr.com) and check out some of the photos of data, instruments, and other things of interest. Or, better yet, why not send an image of your own to the flickr pool. All we ask is that it be flow cytometry related. If you want to send your image, simply compose an email to whose60which@photos.flickr.com. The Subject line of the email will be the photo's title, and the body of the email will be a description of the photo (make sure to remove your signature so it doesn't become part of the description). Then just attach the image, and send it along. It will be posted to the flickr pool, and randomly displayed on the facility's home page, http://ucflow.uchicago.edu

Where would we be without GFP?

In light of the yesterday's announcement of the 2008 Nobel Prize in Chemistry, I'd like to take a minute to demonstrate the importance of this work. The prize will be shared among Osamu Shimomura, Martin Chalfie, and Roger Tsien for the discovery and development of green fluorescent protein, GFP. Yep, that's right, good ole GFP finally getting the props it deserves. Truly, where would science and discovery be without GFP? I estimate that at least 50% of the cell sorting done in our facility is for GFP or its analogs (CFP, RFP, YFP, etc..), and I'm sure that scenario is repeated in every major Institution with a Flow Core. Even in simple flow cytometry analyses, GFP is many times one of the colors people typically look at. If you do a Pubmed search for keyword GFP, you'll get over 15,000 hits, and GFP hasn't even been around THAT long. I won't go into the history of GFP here, but if you're interested, just check out the wikipedia entry. If you're at all familiar with using GFP, then you undoubtedly have heard of or read papers by Roger Tsien, who not only did the early work on using GFP to label proteins in living cells, but has gone on to develop a whole slew of variants. These variants have different spectral properties and expression rates and stabilities, but they all pretty much work the same way. With the addition of many distinct fluorescent proteins, investigators now can label 3 or 4, or more proteins simulataneously in living cells. So, congratulations Shimomura, Chalfie, Tsien, and, of course, GFP.

eBiosciences New Fluorochrome Options = Repackaged Qdots

eBiosciences (www.ebioscience.com) launched a new set of fluorochrome options for their antibody line. They're calling these fluorochromes, eFluor(TM), and they'll be directly coupled to select antibodies immediately. If you're familiar with the Qdot technology currently offered by Invitrogen (www.invitrogen.com), then you're already familiar with eFluor; A nanocrystal core of Cadmium, Zinc, and Selenium with a biologically active coating to allow antibodies/proteins to stick to it. Even though this is simply a repackaging of an already established technology, it will be a great addition to flow cytometry and imaging cytometry. The ability to combine eBioscience's great line of antibodies with the Qdot technology will make using these things more of a reality. Invitrogen has had a pretty limited stock of directly conjugated antibodies, and hopefully eBioscience can expedite this process. The main advantage of these fluors is the ability to use multiple nanocrystals together using a single 405nm or 355nm laser without a ton of compensation. The emission spectra of these nanocrystals are so tight that it allows you to pack them close together without overlap. Also, by putting some of your antibodies in the UV or Violet channels, it frees up your visible laser channels for other, sometimes more important antibodies. To find out more about eFluor, as well as look at what's available so far, click on over to http://www.ebioscience.com/ebioscience/efluornanocrystals.asp for more information.

FlowJo Mac v.8.8 New Features

FINALLY!!! This has been on my list of FlowJo improvements for a long time. One of the features I like best on the DiVa 6 software is the ability to bend quadrant lines to account for the spread that is revealed after you compensate. The positive fraction is always a bit higher than the negative fraction. Therefore, it only makes sense that the line you draw to differentiate between single positives and double positives should have a positive slope to it. The slope of the line will depend on the amount of spillover and the spread that ensues. It was nice being able to easily match that slope with a set of quadrants in DiVa, but that wouldn't translate into FlowJo, so you were forced to create individual polygons for each of the populations. And, when you attempted to do this, it was nearly impossible to get all the gates to line up without any whitespace inbetween (more about that in a minute). But now, FlowJo has introduced in the Mac version 8.8 (go figure, the Windows version falls behind again) 'Spider Gates'. Just option-click the center point of the quads and move to bend the quad lines. Wonderful! My only complaint in actually using this is that after you convert your standard quad gates into a spider gate, you can no longer simply grab the center point and move all 4 quads at the same time. You need to do a select all gates function (cmd+a) and then grab the center. A small price to pay, I think, but hey guys, if you're listening, that'd be nice. The second new gating function is what they call molded gates. This gets back to the point of not being able to get all my polygons to match up exactly with no whitespace and no overlap. Now, if you select a bunch of gates (shift-click them, or cmd+a to select all of them) you can hit cmd+opt+m to 'Mold' the gates together. The couple of tries I've made thus far have worked really well. The gates must be 'nearby' meaning that you'll have to spend some time (albeit, much less) to get the gates close, but then, voila! it just works. If you have any questions/problems, send me an email, or submit your bugs directly to folks at FlowJo.

Regional Flow and Imaging Meeting - GLIIFCA

I'd like to extend an invitation to a really great regional flow cytometry and imaging meeting that focuses not just on flow/imaging technology, but also application innovation and clinical topics as well. Attached is a flyer for the Great Lakes International Imaging and Flow Cytometry Association meeting September 19 - 21, 2008 in Milwaukee Wisconsin. It's close, it's cheap, and you'll get the opportunity to hang out with the flow lab!!!

Bring a poster to the meeting and you're eligible for a $100 stipend to help defray travel/hotel costs. You could also be awarded $150 for being selected as one of the most outstanding poster...like David was in 2006!

Contact the flow lab for more details, consult the attached flyer, or go to www.gliifca.org That's GLIIFCA with 2 I's because we're International (Yeah Canada!).

Students/youngsters may especially appreciate the opportunity at this casual/non-threatening atmosphere to practice your presentation skills.

The trouble with Filters

We've recently gone through some filter issues on the new LSRII-B. It turns out 2 of the filters pre-installed on our LSRII were bad. The two in question are the Alexa 700 filter and the Pac Orange. The Alexa 700 filter (720/40) was a non-standard diameter filter (probably 12mm diameter or so), and when the filter was in place, it would tip forward a bit since it was not snug in the filter holder. This allowed stray laser light or ambient light to get in and swamp the detector. The solution? Simply putting in a standard, 1 inch filter at 730/45. This should now allow for true, 3-color detection off the red laser. The Pac Orange filter was just a bad filter...sometimes you get a bad coating. We received 3 filters to try in its replacement; a 560/40, 550/40, and a 565/30. The winner will be chosen empirically by looking at not just how much PacOrange it collects, but how much Qdot 605 and PacBlue is excludes. If you want to give the different filters a try yourself, send me a note and we'll take a look together.

Next up to tackle on the LSRII-B...PE optimization. The problem with PE on the LSRII-B is the fact that the laser line exciting PE, 561nm, is so close to the emission of PE that there's a propensity to swamp the detector with laser light. I'll be testing a few different filters to see where we can get optimal PE detection without an increase in background from laser light. The PE-tandems, on the other hand, are absolutely fabulous off that laser line. No increase in background, and screaming bright fluorescence... Enjoy!

Is printing hard copies really necessary?

I honestly cannot remember the last time I printed out a hard copy of my flow data. At the end of each flowjo analysis and batch creation, I simply "print" a pdf of the output and save that into my electronic "notebook". The flow lab has recently been going through A LOT of printer ink and paper, and methinks there is a ton of frivolous printing going on. We have slowly been getting rid of printers and only using them for sorting snapshots. You may notice that there isn't even a printer available when you're on the analysis workstations. This is not a computer issue, it's an effort to reduce unnecessary printing. We recommend you make a pdf of your flowjo output, take it back to your lab, and if you really must, print it out there. If you really, absolutely, must print something in the flow lab, ask us and we'll allow it on a limited basis. If you have any questions about doing this in flowjo, just ask and we'll be happy to show you.

Flow Cytometry Data is Beautiful

So, why not make your latest flow analysis into a work of art? Read on for info regarding the "Science in Art" exhibit, or visit uchisciart.org for more details.

Science in Art 2008 Issues Call for Art
Science in Art, a juried art exhibit that features art from scientist-artists from The University of Chicago, Argonne and Fermi National Laboratories and Chicago artists whose subject is science, is accepting art submissions for the Science in Art exhibit 2008 and will accept submissions through Friday, August 22, 2008. The exhibit was developed in response to the need for educating the public about the process, challenges and benefits of science and technology.
Science in Art uses art as a vehicle for creating connections between scientists and non-scientists. As science and technology advance the need for public awareness and understanding increases. The success of science depends largely on public support, therefore communication between scientists and the public is critical. This exhibit will help to translate the language of science for the general public and to push forward the boundaries of human enlightenment. Moreover, it will highlight cross-disciplinary connections in the development, expression and exploration of novel ideas.
Art media accepted for submission include photography, drawing, painting, sculpture, mixed-media, videography and music. Science in Art organizers and a distinguished panel of jurors will select art for the exhibition based on three criteria: (i) how the artwork relates (abstractly or literally) to science (ii) quality of execution (iii) How the artwork goes beyond the mere depiction of the scientific phenomenon.
The Science in Art exhibit will take place at The University of Chicago Gordon Center for Integrative Science (GCIS) Oct. 10-Dec. 13 with an accompanying opening reception on Oct. 10.
Complete entry information, including an application for submitting art, can be found at: uchisciart.org, or by contacting Rebecca Ayers (rayers@uchicago.edu; (608)345-1321.

'Not all DI water are alike' or 'Why the Aria Broke.'

Symptoms: Anything we put through the FACSAria, came out dead. Not like exploded, obliterated dead, just permeable to trypan blue, not-able-to grow, dead. Secondly, tandems looked weird. For example, PE-Cy7 would have a PE-Cy7 positive population, but then it would also have a PE positive population, as if the Cy7 portion of the tandem was getting quenched. However, the PECy7 antibody was fine since it looked normal on the MoFlo.

Troubleshooting: Obviously, the 1st thing to check is the buffer. So, I collected some PBS exiting the Aria's flow cell, mixed that buffer with some cells, and ran them on the instrument and checked them under the microscope. They were fine, suggesting the buffer itself was not killing the cells. I then turned to the (HPLC) valve that controls sample uptake. I disconnected the sample line, after the valve, but before the flow cell, collected some cells, and then reran them. They were fine again, suggesting that it's not the HPLC valve that is killing the cells. So, it seems like the cells have to pass through the flow cell in order to die. On the flow cell, there are wires attached to provide the charging of the stream (required for sorting). So, I disconnected all the charging wires and tried again...still dead. Maybe the lasers are frying the cells. Turned the lasers off...still dead. What to do next?

Placing a volt meter on the metal portions of the flow cell, we discovered a 0.5V charge present. So, perhaps if we grounded the flow cell with an external copper wire attached to a grounding source (in this case, an electrical box behind the Aria) we could keep the cells alive...no luck!

Then, it hit me like a ton of bricks. For about a week now, our Millipore Biocel Water Deionizer was down, so I was getting deionized water from the Monoclonal Facility. Now, the monoclonal facility uses this water for all their tissue culture work, so it is definitely a good source of H2O. The output of water was 'suppose' to be comparable. But then I thought, if the problem really is an electrostatic charge in the flow cell, and, if the new water source is not deionized to the same degree as our original water source, the electrostatic charge could be passing through the buffer, directly to the cells and basically shocking them. Pretty much like an aggressive electroporation.

All the pieces were coming together. It makes sense. This could also explain the PECy7 issue. The extra electrostatic charge was messing with the fluorescence of the fluorochromes as well. So, the obvious next step was to use water from a Biocel Millipore unit. As soon as I did that, all was fine.

So, in summary, buffer made with water from this specific Millipore system, passing through the flow cell on the Aria, with cells present shocked the cells and killed them. Buffer made with water from the Millipore Biocel system..fine. I never found out from BD if that electrostatic charge is suppose to be there or not, but it seems like properly deionized water is required to protect the cells from this charge.

All is well now. Happy sorting!

The new flow cytometry Zune?

So, not really flow cytometry news, but a mockup of the soon-to-be-released special edition, "Joy Division" zune struck me as oddly familiar. The etching on the back. Anyone see a bunch of overlaid flow histograms ala Flowjo? Flow cytometry is now influencing pop culture, or at least pop culture from the late 1970s!

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!

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!