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.
Saturday, May 22, 2010
Qdots: they're not just for lighting up your antibodies any more
Get this; a company (spun-off from Fujitsu) has successfully made a green emitting laser using Quantum Dots. The company, called QD Laser, Inc. (how original) created a quantum dot semiconductor crystal that emits at 1064nm. A quick frequency doubling yields the 532nm laser line we all know and love. Now, they certainly aren't making this for the biomedical research crowd, but hey none of the tech we use today was developed for flow cytometry. It's the trickle down effect that gives us all the great gems. So, you're probably saying, who cares, a 532nm laser, we have a ton of them. Well, besides this being made from a Qdot, it also is super low-power consuming, and it is tiny; see the pic below. QD Laser envisions these lasers along with already available red and blue lasers as the projection source of an RGB miniature projector that can be embedded in smartphones. However, I'm envisioning these lasers on my handheld cytometer.
Wednesday, May 19, 2010
HyperCyt Demo 5/25, 5/26
We will be demonstrating a new 96/384 well sampler on the LSRII-Orange next week. To introduce the platform, we will host a seminar on Tuesday, 5/25 at 10AM in Cummings Room 119. On Wednesday the 26th, we are looking for people who wish to try out the sampler using their own samples. Since this unit will be installed on our LSRII-Orange, please be aware of the lasers/filters on that specific instrument. Here's a few stats on this system. More info can be found on their web site @: http://intellicyt.com/products_hypercyt.php
-Max speed: Full 96 well plate in 2.5 minutes, Full 384 well plate in 10 minutes
-Sample Volume: as little as 2ul with zero dead volume, and as much as a full well
-Can be connected to a variety of cytometers
-Carryover in High Throughput Mode can be 1-2%, but inter-well washes decreases carryover to less than 1%
If anyone else out there has one of these, I'd be interested in your thoughts as well. We've used the BD HTS system on a FACSCalibur with not-so-great results, so are looking for something more high throughput, and more stable.
-Max speed: Full 96 well plate in 2.5 minutes, Full 384 well plate in 10 minutes
-Sample Volume: as little as 2ul with zero dead volume, and as much as a full well
-Can be connected to a variety of cytometers
-Carryover in High Throughput Mode can be 1-2%, but inter-well washes decreases carryover to less than 1%
If anyone else out there has one of these, I'd be interested in your thoughts as well. We've used the BD HTS system on a FACSCalibur with not-so-great results, so are looking for something more high throughput, and more stable.
Tuesday, May 11, 2010
CYTO 2010
Here in Seattle at the CYTO 2010 meeting (the new branding of ISAC, for those unaware). It has been a very typical ISAC, content-wise, but a huge success, location-wise. Seattle has turned out to be a pretty ideal site for one of these meetings. We're having the meeting at the Washington State Trade and Convention Center in downtown Seattle. Being from Chicago, it sort of feels like home, but really, really condensed into about a 10-block radius. Literally, everything you need is in walking distance. So, Seattle definitely gets a thumbs-up from me.
As far as the meeting content, the 'hot' technology is definitely the CyTOF from DVS Sciences. We first saw this instrument at last year's GLIIFCA meeting, but there are now many good examples of the technology in the field...and it looks really nice. As you can probably gather from the name, it's a mass spec instrument that is being used to analyze cells. How? Pretty simply: you basically take traditional antibodies, and instead of coupling them to fluors, like FITC and PE, you couple them to mass spec-type stable isotopes. Then you load your 'stained' sample into the CyTOF, and ituses a laser to basically vaporizes and ionizes the compound in an Inductively Coupled Plasma (ICP-Mass Spec, not Maldi-Tof Mass Spec) yielding a specific signature. Since there are around 100 isotopes available for these types of applications, AND, the resolution between isotope detection, you can basically do a 50 'color' experiment without compensation. So, now, you can do huge multiplexed assays with really, really, good resolution. There are even examples of doing a luminex type assay on this. If you load the beads with a few different combinations of different isotopes, you can easily create a 1,000,000 plex luminex assay! I see an S-10 application in my future!
I'll update with more info again soon, including info about our posters!
As far as the meeting content, the 'hot' technology is definitely the CyTOF from DVS Sciences. We first saw this instrument at last year's GLIIFCA meeting, but there are now many good examples of the technology in the field...and it looks really nice. As you can probably gather from the name, it's a mass spec instrument that is being used to analyze cells. How? Pretty simply: you basically take traditional antibodies, and instead of coupling them to fluors, like FITC and PE, you couple them to mass spec-type stable isotopes. Then you load your 'stained' sample into the CyTOF, and it
I'll update with more info again soon, including info about our posters!
Tuesday, April 20, 2010
iCyt Acquired by Sony: FCM controlled by PSP Motion?
TVs, PSPs, PMPs, Laptops, and now Flow Cytometers? This seems like something out of left field, but Sony recently acquired University of Illinois start-up company iCyt. For those of you not familiar with iCyt, they are developers of the 4-headed sorting monster, the Reflection, and more recently, the Synergy (a sorter/analyzer hybrid), and now the Eclipse (a bench-top analyzer capable of doing particle sizing, absolute counts, multi-well sampling, and multi-color analysis). iCyt has certainly made a name for itself by bringing new and exciting technology to a field overpopulated with BD instruments and clones of BD instruments, and it looks like companies like Sony have taken notice. There was no way iCyt was selling enough instruments, and generating enough capital to do the development it wanted, so this acquisition is probably the best thing that could have happened to them. Gary Durack, founded and President of iCyt (and the former Director of the flow core at U of I) had this to say about the new acquisition, "As a Sony company, iCyt will be able to leverage Sony’s global resources to deliver a variety of innovative solutions to the cell analysis market. This is truly a win for iCyt, its customers, and all who will benefit from these advances." Sony desperately wants to enter the technology side of biomedical research where they feel they can make an immediate impact due to their $79 billion in annual revenue. Keiji Kimura, EVP of Sony had this to say: "iCyt’s experience and technologies will be valuable assets for Sony as it expands into this new business domain. We are confident that this acquisition will accelerate the development of Sony’s flow cytometry business by combining Sony’s expertise in the manufacturing of consumer products with the technological assets of iCyt."
I cannot wait to see the 1st flow cytometer controlled by the PSP motion controller. I mean, it had to happen sooner or later. Kids today (allow me to put on my "old man" hat for a minute) expect everything to work like a video game/social network. So why can't flow cytometers, and for that matter, all biomedical research technologies, operate like every other modern technology. Isn't scientific collaboration just a guise for social networking? Where is the Facebook of science? Why can I not run a flow cytometer with 5 of my "friends" (colleagues-represented by their avatar on screen) assisting me in picking out the important pieces of data? I know some of these things exist in some ways, but if it's not integrating seamlessly into the technology, no one will use it. This is where a company like Sony can come in and make lots of waves. They already know how to do all this stuff; think PSP marketplace, but for flow reagents, or analysis scripts, or cool software hacks. 3rd party, open-source geeks (me included) will jump all over this stuff.
One other area of importance is the whole idea of low-cost CD4 counts for areas of the developing world afflicted by HIV/AIDS. I know this is something that Gary Durack is already pursuing (see Cytometry for Life), perhaps now with a global company with lots of money and lots of influence around the world, a new integrated system that can truly allow for low-cost CD4 counts can be developed. I can't wait to see what comes out of this new marriage.
I cannot wait to see the 1st flow cytometer controlled by the PSP motion controller. I mean, it had to happen sooner or later. Kids today (allow me to put on my "old man" hat for a minute) expect everything to work like a video game/social network. So why can't flow cytometers, and for that matter, all biomedical research technologies, operate like every other modern technology. Isn't scientific collaboration just a guise for social networking? Where is the Facebook of science? Why can I not run a flow cytometer with 5 of my "friends" (colleagues-represented by their avatar on screen) assisting me in picking out the important pieces of data? I know some of these things exist in some ways, but if it's not integrating seamlessly into the technology, no one will use it. This is where a company like Sony can come in and make lots of waves. They already know how to do all this stuff; think PSP marketplace, but for flow reagents, or analysis scripts, or cool software hacks. 3rd party, open-source geeks (me included) will jump all over this stuff.
One other area of importance is the whole idea of low-cost CD4 counts for areas of the developing world afflicted by HIV/AIDS. I know this is something that Gary Durack is already pursuing (see Cytometry for Life), perhaps now with a global company with lots of money and lots of influence around the world, a new integrated system that can truly allow for low-cost CD4 counts can be developed. I can't wait to see what comes out of this new marriage.
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