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.