Tuesday, October 23, 2007

Remote-Controlled Flow Cytometry Part 2

I wrote previously about a new tool the flow lab employs called LogMeIn (see previous post here). This utility is great, but what happens if the instrument you want to control through the logmein web interface is off? Well you'd want to be able to remotely power-on the equipment from a remote location. After doing a bunch of searches on this topic, I can up with a remote power controller that has it own ip address. The box I ended up purchasing is called iboot. You basically plug the instrument into the iboot box, then plug the box into a wall outlet and a network jack. You type in the url of the box into your favorite browser, and a crude power on/ power off interface appears. Of course, it's password protected, so only you will be able to power your instrument on and off. We started using this on our FACSAria and we really enjoy it. Now, from my house, I can power on the instrumet through iboot, open up the software through logmein, do a fluidics startup, start the stream and then drive in to work. By the time I get in to work, the instrument is warmed up, and the fluidics have stabilized. This means that we don't have to waste time in the morning setting up when we can be sorting. It now takes about 5 minutes to prepare for the 1st sort of the day, instead of 30 minutes previously.

Monday, October 22, 2007

Culturing Cells Post Sort

For anyone doing sorting applications, I'd like to clear up a common misconception.  Just for the record, cell sorters are not sterile.  The instrumentation is not housed in a biosafety cabinet or hood, and therefore it is not possible to sort in a STERILE environment.  The cells sorters are operated under asceptic techniques that minimize the potential for contamination.  The sheath fluid (PBS) passes through a 0.22um filter that is changed every few months, and therefore, the sheath fluid is clean.  Before every sort, a fresh aliquot of 10% bleach is run through the sample introduction probe to ensure there is no contaminants in that line.  However, tubes are opened in a non-sterile environment, and collection tubes remain open for, sometimes, hours in a non-sterile environment.  The only way to combat potential contamination is to use antibiotics in your culture post sort for a limited time.  Since many bacterial strains are resistant to pen/strep, we recommend using Gentamicin.  A wide variety of concentrations using Gentamicin in tissue culture have been proposed, anywhere from 10ug/ml up to 2000ug/ml.  We propose using a higher concentration for a period of 3 days post sort to kill any low level contamination that may be present in the culture.  A concentration of 100ug/ml in tissue culture media should be sufficient.  Gentamicin is available from Sigma-Aldrich as their Hybri-Max, 50mg/mL, sterile filtered solution, which can be added as 1ml to a 0.5L bottle of media.  We are continually evaluating methods used in the facility for decontaminating the instrument and the environment around the instrument.  If you have any questions or suggestions, please feel free to send them my way.

Friday, October 12, 2007

Welcome to the new flow web site

The web site for the University of Chicago Flow Cytometry Core Facility has underwent a complete renovation. We're hosted on a new server, updated our url, and rewrote the entire site. I'll take a few moments to walk you through some of the features of this site, but in case you can't find something that you use to be able to see on the old site, we'll leave the old one active for a while. To go to the old site now, click here

The section you're reading now is the recently released flow cytometry blog (ucflow). The center part of the home page will display a portion of the 5 most recent postings to the blog. To read more, or read the archives, you can reach the full blog at http://ucflow.blogspot.com/

There is a main navigation bar at the top, which allows you to jump to the major divisions of the site, Instruments, Services, etc... Next, the left-hand navigation list will change depending on which major section you currently are on, and will display context-sensitive information accordingly. On the right are quick links to commonly used areas of the site, especially links to the analyzer and sorter schedules. These links bring you directly into the read-only version of the scheduler. To actually schedule something, you'll need to log in.

As usual, the site is a work-in-progress, so please be patient. We'll try to update unfinished pages as soon as we can, but if there is something you'd like to add, please send us an email and we'll try to get to it. If you want to send an email to the general flow lab, you can email ucflow@gmail.com otherwise, visit the contact page to send an email to a specific person.

Thursday, October 4, 2007

Using a green laser pointer on a flow cytometer!

Well, the folks at Los Alamos National Labs (nfcr.lanl.gov) never cease to amaze me.  They outfitted one of their slow flow flow cytometers with an ordinary $160 green laser pointer (2.4mW at 532nm), and it works...well, sort or. 

Don't go throwing away your 200mW DPSS 532nm lasers just yet.  I don't know if you caught this, but the "garage-built" flow cytometer this laser is mounted on is a SLOOOOOOW flow cytometer.  For comparison sake, one of the fastest flow cyotometers, the MoFlo (www.dakousa.com) has an illumination time frame of about 1.2us, the slow flow instrument has to have an illumination time frame of at least 25us (~20-fold increase).  Most conventional bench-top cytometers are somewhere between 1us and 4us windows.  From a practical standpoint, the larger the illumination window, the slower your event rate has to be in order to avoid having multiple cells in the illumination area at one point.  So, if you typically run at max 20,000 cells/second on a conventional flow cytometer, you're stuck at 1000 cells/second on the slow flow cytometer.  The nice thing about the slow flow instrument is that since there is a longer exposure time to the laser light, fluorochromes can be excited multiple times, and the fluorescence output increases dramatically.  This is why they can report detection threshold values of ~52 MESF in the PE channel on this instrument.  Not bad.  Although marketing materials from commercial vendors publish outrageous values for the detection threshold, I've never really seen any much below 50MESF, so I'd have to say this is probably more sesitive than any instrument around.  The practical implications of such an instrument, as you could imagine, is the low cost of production for such a cytometer.  Lasers on any other instrument are $10K or more, but for a $160 laser and great sensitivity, I'd be willing to run at a slow rate.  Their findings were published in Cytometry Part A. Habbersett, R. C., Evaluation of a Green Laser Pointer for Flow Cytometry., Cytometry Part A, Volume 71A, Issue 10, p809-817. 

Monday, October 1, 2007

GLIIFCA 2007 Wrap-up

Another informative and successful meeting of the Great Lakes International Imaging and Flow Cytometry Association (GLIIFCA) can be sent to the history books.  UCFlow's poster on our new tools for remote management/troubleshooting/teaching of satellite facility instruments was a hit.  We got lots of traffic, and many laboratories will be trying out the methods soon.  If we get word back, we'll send out an update.

As far as tech that was presented, there wasn't too much "cutting-edge" stuff.  No new instruments (except for the MoFlo XDP, which has been out for a few months now).  The one thing that was new to me at least, was Invitrogen's Click-IT Edu kit for looking at s-phase without the harsh denaturing step associated with BrdU staining.  This seems like a really good alternative to BrdU or tritiated thymidine incorporation.  It basically uses a Uracil analog similar to BrdU and the resulting fluorescence can be pretty much any of the Invitrogen flavors (Alexa Dyes, Pac Blue, etc...) making multiparametric analysis much easier.  It's nearly $9/test (OUCH!!!), but may be worth it, especially if you're doing some complex multicolor stuff.  

I got a chance to check out the Accuri C6 flow cytometer (www.accuricytometer.com).  I wasn't that impressed.  Basically, it's suppose to be a routine, 4-color (FITC, PE, PerCP, APC), 2 laser (488nm, 635nm) basic flow cytometer that costs around $30K.  The price may be right for lots of people, but my major concern is sensitivity.  I think most people who buy this won't use it as it's suppose to be used (a routine, quick, screening tool for well established bright antibodies).  They'll try to run everything on it, and be disappointed.   The gold standard spec, FITC MESF detection threshold is pretty high (<750mesf) compared to a Calibur (<250MESF)

Lastly, there seems to be a paradigm-shift trying to emerge in the analysis of flow cytometry data.  The problem this shift is attempting to solve is firstly, the subjectivity involved in the gating process, and secondly, the shear complexity of analyzing an N-color experiment in N dimensions.  The tools (see verity software www.vsh.com or cira discovery www.ciradiscovery.com) attempt to reduce complex data to a few plots that could be understood by a non-cytometry scientist.  The hierarchical bivariate gating strategy currently employed by users may be on its way out if tools like these are developed further, but only time will tell.  My take:  It's gonna be tough to pull people away from their dot plots and polygons, and I can really only see this catching on in the pharma sector where high throughput is a necessity.  It may be overkill for the run-of-the-mill academic scientist, but who knows, it will definitely be an educational process.