ISX Fluorescence Intensity versus FCM

One of the first questions people ask me about the ISX is how does the fluorescence intensity compare to a standard flow cytometer (FCM). In a quick attempt to partially answer that question, I ran some BD Calibrite beads on our LSRII-Orange and our ISX, and have posted the results in the attached slide. I ran PerCP beads in hopes of demonstrating that the high laser power and long illumination times on the ISX would photobleach the signal (which it did). I also ran FITC, PE, and APC beads as sort of a general comparison of commonly used fluors. The results are fairly nice. FITC is nearly identical on the 2 instruments, PE was brighter on the FCM (probably due to filter ranges), and APC was really bright on the ISX (due to the zero background fluorescence in this channel). The other thing to test was how well the IDEAS software compensated the spillover. For FITC and PE, it was fine. However, because the PerCP was so dim in the PerCP channel, (APC was actually brighter in the PerCP channel than PerCP was) compensating those two took quite a bit of trial and error on my part. I manually adjusted the comp values until it looked acceptable. The resulting plot pretty much explains it all. You'd be hard pressed to easily identify APC, PerCP double positives in this example (luckily there were none, so no problem). Anyway, just wanted to give you a quick peak into how things may match up when trying to set up your color choices for the ISX.

Biolegend's Kelly Lundsten in tha house

We are hosting a seminar on multicolor cytometry next Thursday and we think it should be an absolute blast. We got fluorescent chemistry specialist Kelly Lundsten from Biolegend to come over and talk about the different factors involved in choosing the right panel of fluorochromes in a flow cytometry experiment. For example, she’ll cover the level of antigen expression, with signal:background of the fluor and detection channel, how to categorize antigens to minimize the need for multiple fluorophore versions of the same antibodies, etc. There will be an example of a 10 color assay optimization and the use of FMO controls for gate placement. She will also cover the ins and outs of the different dye chemistries too, organic dyes, proteins and nanocrystals from both a theoretical and practical perspective. Seriously, ask her about nanocrystals. Then try to interrupt her. Fun.

The seminar will be held on:

Thursday April 22nd 2h30PM to 4PM
KCBD Auditorium (room 1103)

Refreshment and snacks will be served.

This should be a very cool talk. Hope to see you there. Let me know if you have any questions.

Easy FTP data Transfer

You will now see an icon on the desktop of each "windows-running" computer that says "Upload data to FTP Server." You can begin using this to transfer your data files from the acquisition computer to the Data Server. You can then log into the FTP server from your desktop computer and download the data files to your PC. Please note that the Data Server is NOT a data storage server, it is simply a means for transferring your data to your computer without having to use removable media (USB drives, CDs, etc...). You can upload your data to the server from the Macs as well, but you will not be using the Fling application. We will just map the directory on the FTP server to an alias on the desktop. Detailed instructions will be mailed to the list, if you do not receive these emails, send us an email directly or call the lab.

Now that's more like it. ISX Data Samples

We've had the opportunity to run a few real experiments on the ISX over the past week or so, and the data we're collecting seem quite interesting. We've performed the following experiments: NFkB translocation, GFP Foci Counting resulting from DNA Damage, Ploidy Analysis using CEN Reference Cells, and Cell Cycle. I've attached a few images of some compiled .ppt slides. They should be pretty self explanatory, but here's a quick run-down.
The 1st slide is looking at Chicken Erythrocyte Nuclei which is typically used as a reference peak when doing DNA ploidy analysis. These nuclei tend to pack closely together yielding discrete peaks at 2N (1 nuclei), 4N (2 nuclei), 6N (3 nuclei) and so on. The one interesting thing to note here is the ability of the ISX to display up to 20N (decaplets) on a linear scale. This gives you an idea of the dynamic range possible with this system. On a traditional Flow Cytometer, you'd only be able to display up to 8N on a linear scale due to the narrow dynamic range.

The second slide is a Nuclear Foci Counting experiment. For this we used the EDF filter (Extended Depth of Field) in order to get all foci in 3 dimensions in focus at the same time. The remarkable thing here is the ability for the IDEAS software to locate and count the number of foci inside the nucleus anywhere from 1 foci up to 25 foci.

The third slide is a simple cell cycle analysis. As you can see this looks much better than my 1st attempt. What's really neat, is that using a few more morphology parameters such as the "centroid" feature, we could easily separate out the late mitotic cells (easily discernible from the images in the figure) from the G2 cells. Of course we could always add in a marker or two to label the phospho-Histones to make that process easier, but you get the idea.

The last slide is of an NFkB tranlocation experiment. The analysis involved here compared the nuclear staining DAPI with the NFkB translocating subunit stained with FITC. If the staining is similar (i.e. translocated NFkB) a higher similarity score would be given, and if the staining is anti-similar (non-translocated NFkB) a low or negative score would be given. In this example we are simply comparing stimulated versus non-stimulated samples demonstrating a higher level of nuclear translocation of NFkB in the stimulated sample.

So, as you can see, very interesting data is being collected. If you have any questions, or want to try something, give us a call. Remember there is NO USAGE FEE on this instrument for a limited time!

It ain't pretty, but it works...

I'll probably look back at this some time very soon and realize how bad this really is, but I figured I'd post it anyway. What we have here is the 1st data file collected on the ISX. I basically took the field service guys' test cells (which happened to be fixed K562s) threw a few microliters of PI on them and stuck it on the instrument. No incubation, no RNAse, no knowledge of how the cells were actually fixed (PFA, Methanol, other), no real clue on how to operate the instrument whatsoever. So, you can sort of understand my mild level of excitement that I actually got a somewhat discernible cell cycle profile. I collected the data using the Inspire software, and the shot it over to the analysis computer with the IDEAS program and played around a bit. I did a few screen grabs and made a layout in paint (that's why the resolution of the images is so poor) and displayed it below. If you squint really hard, you can see a G1, G2 and S-phase profile, and then I grabbed a representative image from those 3 groups to confirm they were actually G2 cells. I was even able to do a pretty good job of excluding aggregates. So there you go folks, it works. I guess we'll keep it.

ImageStream. Installed.

It seems like years ago we were racing to put together our S-10 to purchase an ImageStream, and yet, it's only been 1 year (almost to the day!). I guess the anticipation has made the wait that much more excruciating. Well, wait no more, the ImageStream is just about installed. The engineers from Amnis arrived on Monday to uncrate and assemble the surprisingly small ImageStream. I should just clarify something right off the bat. The instrument we have is the current generation ImageStream X (not sure what the X means, maybe it stands for X-treme - remember that tag line from the MoFlo XDP...a little dramatic if you ask me). Anyway, I've become fond of the abbreviation, ISX, so that's what I'll be referring to it as. Out ISX has quite the spec sheet; 4 lasers (405nm, 488nm, 561nm, 658nm), two 6-channel CCDs (up to 10 colors, Brightfield and Darkfield), the multimag (magnification from 20X - 60X), and the EDF (extended depth of field). I'm very anxious to get my hands on it. If you'd like to see it in its pre-Ryan Duggan, pristine state, you'd better stop by and take a look at it soon, because one I'm let loose, I have a hard time leaving things alone. I'll try not to break anything for at least a few weeks. We'll be getting our training next week, and then we'll probably start opening it up to users the following week (April 5th). If you think you're ready to go with something on this instrument right away, please contact me and I'll get you on the list ahead of posting the instrument on the scheduler. If you have no idea what the ISX is, check it out here.

Data Transfer post USB

I'm over USB for data transfer. There's the possibility of losing the drive altogether or it becoming corrupt, or in the case of recent happenings, it could get infected with a virus and then transmit said virus to every computer you plug it into. CDs, DVDs, BDs, may not be as hard to use, but it's still physical media that you need to label and store (away from heat and light, hopefully). So, we got to thinking, why can't we do without physical media altogether. We, in the flow lab IM or email ourselves data all the time. Sure there's a limit on the total byte size, but many of our experiments involve small numbers of tubes/small numbers of data points. Enter, the ImageStream. The data from the ImageStream (btw, it's being installed this week...more on that later) is going to be much larger, so when I was writing the grant, I put in some funds to get a full-out server that would allow us to at least FTP data to and from. If I'm already setting that up for the ImageStream, we should be able to use it for all the instruments, and manage data sans removable media across-the-board. So, over the next few weeks, we will be implementing this FTP to Server strategy on all the instruments. The LSRIIs were the test case, and they seem to be working well. I'll be outlining some detailed instructions in the next posts, but basically once you export your FCS files in the normal fashion, just zip the folder and drag it onto the upload shortcut icon the instrument desktop. Then, go to your computer and download your zipped folder of data. For the most part it should be pretty painless. The only difficult part is having an FTP client on your computer that is fairly easy to use. We will be recommending the free, Filezilla program. It runs on Mac, Windows, and Linux, so we can pretty much have 1 set of instructions for everyone, and will be able to troubleshoot, to some degree, problems you may have. For some of you who use other cores on campus (e.g. DNA Sequencing Core) this should be very familiar to you already. If you don't want to use a standalone FTP app, you can set up a handy "network place" on windows, or directly connect to the server on a mac (general instructions to follow). There's always the possibility of using a browser for this, but I'm not a fan, so won't even give instructions for doing that. I will send an email out to the list with the details of the server (server path, usernames, passwords, etc...), and then check back here to get instructions on how to proceed. If you're familiar with using FTP, then the server path, username, and password should be all you need.