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!