Flowcast? What's a Flowcast?

Flowcast is a derivation of the now popular term, screencast, which in and of itself is a derivation of the term broadcast. Now, although there really isn't any "broadcasting" of the screen in a screencast, the term simply refers to a tutorial or guide that uses a screen capturing program so you can see exactly what's going on and follow along easily. These screencasts are then broadcast on the internet for all to see. These types of tutorials work (for most) much better than simply reading through a protocol or document of directions. Flowcasts, therefore, are screencasts relating specifically to flow cytometry. To this avail, the flow cytometry facility has begun producing short (<10 minute) tutorials on whatever strikes us at the moment. We envision having mostly tutorials on how to do certain tasks in FlowJo and FACSDiVa, because that's what we mostly work with, and that's what most of the questions that are asked of us deal with. You'll see in the right hand quick links bar of our homepage (ucflow.uchicago.edu) a link for Flowcast. Check it out and let us know what you think. We would also like to encourage anyone who has an idea or trick that they like to use, let us know, and we'll make a flowcast out of it. The easiest way to contact us is at the following email address: ucflow@gmail.com

The significance of Significance

Did you ever wonder why we always collect 10,000 events in our files? Why not collect less? Or more? Well, to answer this question, you'll need to take a step back and stretch your brain to remember that pesky stat class you took as an undergraduate. You may remember that French mathematician with the funny name, Poisson. Well Poisson's distribution described the probability of a number of events occuring in a fixed period of time IF the events occur with a known average rate AND are independent of the time since the last event (don't believe me, check for yourself on Wikipedia). With that in mind, we'll need to assume that the events going through the flow cytometer pass through the instrument at a given and stable rate in order to allow us to apply the Poisson distribution to our flow data. Not a very difficult assumption knowing that our sample is pretty evenly distributed in a fluid, and that fluid passes through the instrument at a given flow rate. So, applying Poisson, the random and discrete occurrences or "arrivals" are the cells passing by the laser intercept, one by one. Also, Poisson tells us that if N events, or "cells", are observed or "collected" then the standard deviation (SD) associated with the collection is the square root of N. Brilliant! Additionally, we can express the SD in a more friendly term, the coefficient of variance (CV), which is 100 x SD/N. The CV is will give us a percent variance of our distribution. Very tight distributions will have a low CV, and very broad distributions will have a large CV.

Now, how is all this pertinent to flow cytometry. Well, lets say you collect your 10,000 cells, and you are analyzing a subset within that 10,000 cells that is at a frequency of 10%. This means, you've collected 1000 cells in your subset. The variance you want to measure and test the significance of is the 1000 cells in your subset, so, Poisson tells us that the SD of this population is equal to the square root of 1000, which is 31.62. If the SD is 31.62, then the CV is 100 x SD/1000 or 3.16%. This means that the accuracy of the frequency you report on the 1000 cells you collect has a variance of 3.16%. If you would like your CV limited down to say, 1% variance, then you would need to collect a data file of 100,000 cells, giving you a subset of 10,000. Given this simple equation, you can calculate ahead of time just how many cells you need to collect in order to achieve a certain amount of variance. A nice table and description is available at the following link from the folks at Cardiff University in the UK. So, in answer to our 1st question, we need to collect as many cells as will allow us to derive conclusions from our data that is backed up by significance.

RoboSep Demo Unit Available in Flow Lab

If you're doing any magnetic separations using beads from StemCell Technologies, or Miltenyi, or even Dynal beads, this unit may be of interest to you. The RoboSep from StemCell Technologies uses a very simple design to automate the pipetting necessary to couple your cells of interest to the paramagnetic beads. Once coupled, the built-in magnet will fractionate the coupled cells and the uncoupled cells allowing you to positively or negatively select your cells of interest. There are 4 magnets in the unit allowing you to fractionate up to 4 samples simultaneously. The touch-screen interface allows easy setup of samples and incubation times. The utility of the "magnetic sorters" can be maximized in the case that you wish to sort cells in the facility. A pre-enrichment of the population of interest can be done magnetically allowing you to decrease the amount of time on the sorters.

During the time that the facility has the demo unit, we will ask anyone interested to please try it out. Our StemCell contact is Rory Connelly (rory@stemcell.com). He has agreed to assist you with getting your samples run on the instrument, and the flow facility will allow you to re-run your samples on the analyzers free-of-charge (to check purity and yield post sort). We would also like to get feedback on the usefullness of such an instrument in the core. Would you realistically travel to the core facility to do your magnetic separations? Does it actually work for your project? This information will be very useful as we evaluate the instrument.

Feel free to contact us in the flow lab as well if you want more information

Amnis ImageStream Update

First of all, thanks to Scott Levy, and Tad George from Amnis for putting together a fine presentation. We'd appreciate it if you could take 30 seconds and fill out a 3 question survey from the flow facility regarding people's impression of the technology. Click here to take the survey.

The flow facility will be attempting to get some investigators to send samples to Amnis and generate some data. This way, you can get first hand information as to the utility of such an instrument. Your samples, your model, your questions. This will greatly help us in determining the likelihood of an instrument like this being used on campus. So, if you think one of your experiments or projects may benefit from this technology and want to try some samples (free of charge, I might add). Then send an email to rduggan@bsd.uchicago.edu, and I'll get you some info on sample prep and whatnot.

Amnis ImageStream Technical Seminar

The Flow Cytometry Facility will be hosting a technical seminar on the (not-so) new technology from Amnis (www.amnis.com) called the ImageStream. It acts very much like a flow cytometer; it has a fluid stream, lasers, detectors, and the such. However, it has one additional feature that makes it a totally different instrument. The detectors are actually CCD cameras, and they collect images of each cell as it passes through the lasers. You also get a bright-field image! This gives whole new meaning to your data since now you have the added power of morphology and fluorescence localization. You may be saying, well, yeah, that's nice, but i already do that in microscopy. The difference here is the power of "flow-like" computation. Standard fluorescence microscopes apply some software to try and deconvolude the data it records to get information like, frequency of a population, or fluorescence intensity measurements. Since these cells flow one-by-one through the sensing portion, there's no need to try and fish out whether what you're seeing is a single cell, or two cells that are really close together. Some of the "killer apps" for this instrument includes Nuclear Translocation, Apoptosis, Phagocytosis, Molecule Trafficking, FISH, and Gene Expression. The familiar dot-plot or histogram data presentation makes analysis very intuitive for a flow cytometrist. For more information regarding the upcoming seminar, please contact Ryan Duggan (rduggan@bsd.uchicago.edu).

Details:
Thursday, November 15, 2007
12:30PM - 1:30PM
BSLC Room 001

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.

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.

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.

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. 

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.

Want the Flow Lab to look over your shoulder?

The flow lab has begun using a web service called LogMeIn (LogMeIn.com) which allows us to remotely log into any of our computers on campus from anywhere a network connection can be had.  What this means is that if you're on, say, the Canto in S319, and you'd like a second opinion on whether or not your voltage settings look ok, just send us an IM (flowhelp) and ask us to log in and take a look.  We will have full control of the computer, so we'll actually be able to manipulate all the controls on the instrument while simultaneously walking you through the steps through IM communication.  Pretty cool, eh? 

GLIIFCA 2007, Windsor Ontario

The flow facility will be attending the annual Great Lakes International Imaging and Flow Cytometry Association (GLIIFCA) meeting this upcoming weekend (9/26/07-9/30/07).  This annual meeting discusses the latest and greatest in technology and applications within the Flow Cytometry field.  Emphasis is placed on technical advancement in Clinical Applications, Research Applications, and Technology.  The flow facility will be presenting an abstract entitled:  "Remote-Controlled Flow
Cytometry: The use of remote access communication tools for distance learning."

A little background about the R409 Canto Computer Issues

The FACScanto Computer in R409 has had a symptomatic problem where it randomly shuts down during acquisition.  It's not that it actually goes through the shut down process, but it appears that the screen and computer just go black!  People have dealt with this by starting up the computer again, and praying that it doesn't happen any more.  Some users have attempted to correlate these findings with the acquistion of large data sets (i.e. acquiring for a long time).  We've, however, seen this happen pretty randomly.  The kicker is that when you check for any errors in the log files, nothing shows up.  Steps we've taken thus far including uninstalling and reinstalling both specific applications (e.g. FACSDiVa) and the OS.  Additionally, we replaced the power supply of the unit, and placed it behind a UPS so that if there were power failures occuring, the UPS would provide some power backup.  It seems none of this has worked, so we are now at the point of more drastic measures.  One option would be to replace the computer.  Sounds easy enough, except for the cost.  These customized computers, which MUST be bought from BD cost upwards of $10K!  So, a purchase such as this is not trivial.  The other option would be to replace the motherboard, which we could probably get for $1K.  Our IS guru is currently looking into options, and we've decided to just put the instrument on the down-list until we find a more permanent solution.  Stay tuned for more details.