Wednesday, June 23, 2010
You mean there's other analysis software besides FlowJo???
Even though a vast majority of users at U of C are FlowJo users, it doesn't mean we ONLY use FlowJo. FlowJo does a really good job with a lot of different flow applications, but there are some things I'd definitely want to change. So, I'm always playing around with new software, as well as revisiting old standbys: FCS Express, VenturiOne, Kaluza, Winlist, to name a few. In addition, there are a bunch of freeware apps that are pretty good as well: WinMDI, Cyflogic, MFI, ANALYSE, IDLYK, and more being developed frequently. Recently I had the opportunity to talk with Kelly Rae Chi from The Scientist Magazine, which resulted in an article featured in the most recent edition. The piece is titled "Let the Data Flow: Rethink your Data Analysis Tools for Flow Cytometry." It's in Volume 24, Issue 6, Page 63.
Wednesday, June 9, 2010
Thursday, June 3, 2010
Intellicyt HyperCyt Impressions
Well, the HyperCyt has come and gone and to sum up my thoughts in a word I can only say, Wow. When it comes to speed, this thing is really fast. I tried to put a bunch of different things through the sampler to try and get it to fail, but it just didn't; big stuff, clumpy stuff, small stuff, dyes, you name it.
Now, let me put things into perspective for you a bit. We run everything in tubes manually, so to run through 96 samples I've been able to achieve a throughput rate of about 40 minutes (collecting a couple of thousand cells per sample). My target for a sampler was to get an equivalent amount and quality of data in less than 20 minutes per plate. By many accounts, this is NOT high throughput. The representative from Intellicyt actually sort of chuckled at me when i was explaining this. His idea of high throughput was collecting 384 well plates in about 6 or 7 minutes. I certainly can appreciate the utility of this unit for something like that, but my standards of data quality are probably a bit more strict when compared to a "screening" assay.
What I was really impressed with was the ability to precisely control every aspect of the sampling process such that you can go really fast, or pretty slow if you wanted to. What I am showing here is a comparison of the same exact plate run at a "high" speed (~11 minutes per 96 well plate) and at a "low" speed (~27 minutes per 96 well plate). It is a simple Annexin V FITC vs. 7-AAD cytotoxicity assay, that is comparing the affects of a drug on the cells. As you can see, there is no appreciable difference in the two, and they both are the same as when I ran these samples manually in a tube.
Other things I ran were two big cell lines (one GFP, one RFP) mixed together to look at the potential increase in coincidence events (double positives) when going at high rates. When I ran the sample manually in a tube, I saw about 0.5% double positive cells. When I ran them through the HyperCyt at the higher flow rate, that double positive population shot up to 1.6%, and when I ran it at the slower flow rate, it was about 1%. The reason this happens, as I'm told, is that when the unit sends the bolus of cells into the cytometer, the cells tend to accumulate at the leading edge of the bolus, so you get a high concentration of cells right in the beginning of collection, thus leading to higher coincidence. In fact when I looked at the formation of coincident events versus time of a single collected sample I saw exactly that, all the double positives were detected in the 1st second or so of collection, then after that, it leveled out to the 0.5% seen while running the samples manually in a tube.
One other thing I was concerned with was the residual dyes left over when rinsing between samples. To test this, I ran alternating samples of PI stained CENs and DAPI stained CENs. I first ran it with no wash to show that the probe itself may be transferring dye from well to well (which it did). Then I ran the samples with a wash in between collection and then there was no increase in PI staining of the DAPI CENs and no increase in DAPI staining of the PI CENs. This, along with multiple other tests run with beads put me at ease as to the capabilities of this instrument as a multi-well plate sampler, not just a high throughput screen tool.
So, will I get one? Likely, but possibly in the form of a Accuri/HyperCyt combo ala what was displayed at the CYTO 2010 meeting. This is a new product from Intellicyt, which they're calling the HTFC (High Throughput Flow Cytometer). Here's a link to the info on that (warning, this is a link to a pdf, so it will automagically start downloading to your computer; not to worry though, it's safe).
Now, let me put things into perspective for you a bit. We run everything in tubes manually, so to run through 96 samples I've been able to achieve a throughput rate of about 40 minutes (collecting a couple of thousand cells per sample). My target for a sampler was to get an equivalent amount and quality of data in less than 20 minutes per plate. By many accounts, this is NOT high throughput. The representative from Intellicyt actually sort of chuckled at me when i was explaining this. His idea of high throughput was collecting 384 well plates in about 6 or 7 minutes. I certainly can appreciate the utility of this unit for something like that, but my standards of data quality are probably a bit more strict when compared to a "screening" assay.
What I was really impressed with was the ability to precisely control every aspect of the sampling process such that you can go really fast, or pretty slow if you wanted to. What I am showing here is a comparison of the same exact plate run at a "high" speed (~11 minutes per 96 well plate) and at a "low" speed (~27 minutes per 96 well plate). It is a simple Annexin V FITC vs. 7-AAD cytotoxicity assay, that is comparing the affects of a drug on the cells. As you can see, there is no appreciable difference in the two, and they both are the same as when I ran these samples manually in a tube.
Other things I ran were two big cell lines (one GFP, one RFP) mixed together to look at the potential increase in coincidence events (double positives) when going at high rates. When I ran the sample manually in a tube, I saw about 0.5% double positive cells. When I ran them through the HyperCyt at the higher flow rate, that double positive population shot up to 1.6%, and when I ran it at the slower flow rate, it was about 1%. The reason this happens, as I'm told, is that when the unit sends the bolus of cells into the cytometer, the cells tend to accumulate at the leading edge of the bolus, so you get a high concentration of cells right in the beginning of collection, thus leading to higher coincidence. In fact when I looked at the formation of coincident events versus time of a single collected sample I saw exactly that, all the double positives were detected in the 1st second or so of collection, then after that, it leveled out to the 0.5% seen while running the samples manually in a tube. One other thing I was concerned with was the residual dyes left over when rinsing between samples. To test this, I ran alternating samples of PI stained CENs and DAPI stained CENs. I first ran it with no wash to show that the probe itself may be transferring dye from well to well (which it did). Then I ran the samples with a wash in between collection and then there was no increase in PI staining of the DAPI CENs and no increase in DAPI staining of the PI CENs. This, along with multiple other tests run with beads put me at ease as to the capabilities of this instrument as a multi-well plate sampler, not just a high throughput screen tool.
So, will I get one? Likely, but possibly in the form of a Accuri/HyperCyt combo ala what was displayed at the CYTO 2010 meeting. This is a new product from Intellicyt, which they're calling the HTFC (High Throughput Flow Cytometer). Here's a link to the info on that (warning, this is a link to a pdf, so it will automagically start downloading to your computer; not to worry though, it's safe).
Core Fair Today!!!!
The Office of Shared Research Facilities is hosting it annual Core Fair today from Noon to 2PM in the GCIS atrium. This is your chance to visit with all of the ~25 different core facilities on campus to find out what's new and exciting in their labs. The flow lab will be present and is eager to share with you our new technology and services. We'll be highlighting the ImageStreamX, of course and all the neat things you can do with that instrument. But, we're also trying to spread the word about our Drop-Off Service and our forthcoming analyzer, the BD LSRFortessa. Last, but by no means least, we'll be letting people know about our exciting news regarding the MoFlo. (Drum roll, please) It has been a long time coming, but we've finally secured some funding to get our MoFlo upgraded to the MoFlo XDP package from Propel Labs. This will definitely breathe new life into our dying sorter.
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