Plant Sample Prep for Nuclei Analysis

We don't have too many Botanists coming through the flow lab doors, but occasionally they'll bring in some electric green looking slurry of stuff and want to analyze DNA content or Mitochondrial Membrane Potential. It's really no different setting these samples up on the instrument than any other animal/human sample. But, when they start to ask me questions on how to optimize their sample prep, I'm of little help. From what I understand, the process is much like dissociating any tissue. You use some mechanical (slicing and dicing with a razor) and enzymatic processes to try and isolate the cells from the leaves. Beyond that basic understanding of what's going on, I have little to offer, which is why I appreciate this troubleshooting guide so much. A fella by the name of Paul Kron, from the Department of Integrative Biology at the University of Guelph (Guelph, Ontario, Canada) sent me his top 10 troubleshooting tips for achieving good quality histograms when analyzing DNA content from plant material. There's probably some things in here you'll think are somewhat elementary, but isn't it always those elementary steps that are lacking when things aren't going quite right? A short, easy-to-read guide like this will ensure you've thought about some of the minutia involved in optimizing your DNA profiles from plant materials. A link to the top 10 can be found here: http://www.uoguelph.ca/~pkron/PaulKron/Top_10_FCM_Tips.html.

Do you Kaluza?

Seems like there's quite a bit of news coming out of Miami these past few months, and that is a good thing. Beckman Coulter (coulterflow.com) has released it's offline analysis tool, Kaluza, whose major feature seems to be speed. I've been playing around with Kaluza for about a month now and so I can share a few thoughts. You're probably well aware of the fact that we at the University of Chicago are a FlowJo shop. Nearly all of our users analyze with FlowJo (others use acquisition programs like CellQuest or FACSDiVa to do analysis). We've looked into other packages in the past (namely FCS Express and VenturiOne) but none were as exhaustive in capabilities as FlowJo, so we felt we were getting the most bang for buck in that case. What this basically means is that I pretty much look at all analysis software through a "FlowJo-colored lens." My impressions therefore, are fairly skewed towards that bias. With that being said, here's my first impressions on the windows-only (so sad), late beta version of Kaluza.

First of all, it is aesthetically pleasing to look at and interact with. They successfully pulled off the minimalist/high-tech look and feel of the software. Getting started with the software is easy enough. You load FCS files, apply analysis plots/regions/stats and that's it. I've loaded small (10K cells, 2 or 3 colors) files as well as quite large (5M cells, 10 colors) files. They initially load about as fast as you'd expect, within a few seconds up to 30 seconds, but once they're loaded, you can make adjustments, move gates around, recalculate stats very quickly. Moving around within the software is very snappy. This is a welcomed change of pace from FlowJo, which feels like an eternity when manipulating things like compensation on many large files at once. The second thing about Kaluza is navigation. Things like tabbed analyses, the radial menu, and 'office-like' ribbon menus make performing simple tasks inside the software, well, simple. There are a few things I'm a bit unclear of at this point. Apparently batching stats and pdfs are possible in the software, but i haven't quite figured that out yet. Perhaps now that it is out of beta, and user manuals are being put together, I'll be able to play around with that more. However, it is definitely not as powerful as the batching options in FlowJo (their words, not mine). Also, being from Coulter, the nomenclature of certain things is definitely derived from the clinical world, which takes some getting use to. Panels, Protocols, Tests, are all used in a strict clinical sense, and don't always translate directly to the research world, so figuring out what is what took a bit of effort on my part. I have a feeling, the 1st adopters will probably be Coulter hardware users and disgruntled FlowJo users. The speed of analysis is a tempting feature for potential converts, but I may hold off until version 1.x or maybe 2.0 and see what happens. Until then, if you're just dying to try something new, let me know. I'll probably load the software on one of our analysis computers so people can play around a bit if they'd like. Or, if you'd like to get a demo copy of your own, visit http://www.coulterflow.com/bciflow/kaluza.php and click on demo request.

UPDATE: (From Ernie Anderson, BC) FYI, batching stat and PDF output is pretty easy. Just load the files you want to process and select them in the analysis list. (The usual Windows SHIFT-click, CTRL-click, CTRL-A shortcuts work here.) When you have more than one analysis selected, the display is replaced by several buttons:

"Export statistics from selected" will produce a single file containing the displayed statistics from each analysis. Make sure you're showing the stats you want to display before you export. If you're not picky it's easy to just select all the plots and turn on all the stats.

"Print all sheets from selected" and "Print report sheets from selected" do what they say. If you want PDF output, choose a PDF printer driver. There's one on the program CD if you don't have one already. (Sorry, you didn't get that with the beta.)