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!