Flow Core Newsletter

July-August 1997

This is what's new in the Flow Core...

The FACSCalibur is available for use. Our Becton-Dickinson FACSCalibur has had all (most?) of the bugs worked out of it and is now being routinely used by several laboratories for three- and four-color analysis of flow samples. As you may recall, this instrument differs from the FACScan in that it has two lasers: a conventional argon laser emitting at 488 nm, for excitation of FITC, PE, PI,etc. (as on the FACScan) and a diode laser emitting at 630 nm. This second laser makes possible simultaneous analysis of four fluorescent parameters (compared to the FAScan which can analyze only three). Thus, FITC, PE and PerCP-conjugated antibodies can be used in conjunction with a fourth red-emitting fluorochrome. Examples include allophycocyanin (APC) and Cy5, both of which can be excited by a diode laser and detected by the new instrument. As mentioned previously, the Flow Core has purchased limited quantities of APC- and Cy5-conjugated secondary antibodies and avidin to introduce users to the possibilities of 4-color phenotyping. We can also help you find commercial sources of APC- and Cy5-conjugated antibodies, and can help you conjugate Cy5 to your own antibodies and proteins. One laboratory (Dr. Lacy’s at Cornell) is currently using the instrument to detect CD3, CD8, b220 and surface Ig simultaneously on transgenic mouse splenocytes (using APC, FITC, PE and PerCP-conjugated antibodies). Contact us for assistance in working out your own protocols.

Improvements to the B-D Vantage. Our five-color high-speed B-D Vantage sorter has been retrofitted with several improvements, including a temperature-controlled sample holder with recirculating water bath and a new Macintosh computer for improved data handling.. In addition, we have acquired a Cytek Time Zero kinetic module, which will allow for analysis of calcium uptake, transmembrane potential, etc.over time. This unit allows the rapid addition of mitogens, drugs, etc. to viable cell samples and provides an air boost to deliver the cells to the laser intercept within 2 seconds of drug addition, allowing us to observe very rapid changes in cell physiology following drug treatment. This unit will be ideal for use with such calcium probes as indo-1 and fluo-3, transmembrane indicators such as BCECF, SNARF, etc. Other possible applications include: looking at the time kinetics of fluorescent drug efflux by cancer cells expressing p-glycoprotein; measurement of mitochondrial potential following drug treatment or induction of apoptosis using rhodamine 123. Talk to us about it.

Flow Core office. The Flow Core now has office space in Room 215 of the HSS Research Building. This room houses all of our flow-related books and reference material, computer software, reprints and supplier information, as well as our Pentium PC with printer. For security reasons this room will usually be locked; talk to me to get access.

Relocation of the Macintosh remote workstations. All of the Macintosh remotes workstations have been relocated from their original locations (3^rd and 4^th Floor of the Research Building) to the ground floor office suite. This move will allow all of the computer to be hooked up to the network, and will make them more secure against possible theft. The ground floor office suite is normally open during business hours (8:00 AM to 6:00 PM). If you need after-hours or weekend access, get a loaner key from me in advance.

In addition, we will be "test-driving" a new flow cytometry analysis program on the ground floor PowerMac. Called "FlowJo", it was developed by the cytometry research group at Stanford and utilizes a unique, logical tree-based approach to analyzing flow data. A fully functional trial version will be available on the PowerMac on the ground floor. Let me know if you are interested and we can try analyzing your data on it; if the overall response is positive, we may purchase a full version of this software.

New computer. The Flow Core has recently acquired a new Pentium 200 MHz PC. This computer can be used by any facility user for the transfer/conversion of FCS 2.0 files from the Mac to PC format, giving you the option of analyzing your data on the PC (including your own PCs - ask us how to do this). This computer can also be used for analysis of flow data, preparation of flow figures for publication and access to flow-related resources on the World Wide Web. We currently have the following flow-related programs on the PC:

• WinList ver. 3.0. A flow analysis program particularly useful for batch analysis of large numbers of files.

• WinMDI ver. 2.5. An freeware flow analysis program written by Dr. Joe Trotter at Scripps. Has nice graphics. You can also get this program for your own PC - ask us how.

• PC-LYSYS ver. 1.0. A Becton-Dickinson program for analyzing flow data on the PC.

• HP-Reader. A program for converting flow data files from HP to PC format. Useful for getting at data stored under the old HP UNIX system (which Becton-Dickinson used to use).

• Cyclred ver. 1.1.1. A DOS-based cell cycle analysis program designed by Drs. James Watson and Michael Ormerod in Great Britain. You can get this program for your own PC, too.

• HP LIF Utilities. A program for converting and reading old HP UNIX flow data files.

In addition, we also have image editing and drawing programs to convert your raw flow data into clear, polished figures for publication. These include:

• Adobe PhotoShop (image editing)
• Corel Photo-Paint (image editing)
• Microsoft PowerPoint (construction of figures and slides)
• Corel Draw (construction of figures and slides)

We also have the following on-disk and CD-ROM resources:

• Linscott’s Directory on Disk 1996-97
• Immunology Methods Manual on CD-ROM (Lefkovitz)
• Immunobiology Bookshelf on CD-ROM (Janeway)
• Sigma Catalog on CD-ROM
• The Purdue Cytometry CD-ROMs volumes 1 and 2 (includes the entire Purdue Cytometry e-mail archives from 1992 to present on CD-ROM)

All users (HSS and outside) can access this computer, but please talk to me before you use it!

Improvements to the Flow Core laboratory. We have rearranged the Flow Core and set up a laboratory bench in Room 312. This space is available to users who need to perform manipulations on their samples immediately prior to analysis. We have also stocked this space with basic laboratory supplies and equipment (pipets and pipetors, plasticware, stir plates, vortexers, etc.) for your use. In addition, this space enables us to assist you in the preparation of fluorochrome-conjugated antibodies and proteins for use in your f low projects. So far, we have assisted several labs in the preparation of simple fluorescein-conjugated antibodies and tracer proteins, as well as more unusual UV-stimulated, blue-emitting coumarin- and Cascade Blue-conjugated antibodies for use as a fifth color on the Vantage. Talk to us about your needs; we already have most of the necessary reagents in-house.

Assistance with preparation of flow data for publication. One of the major problems with preparing flow data for publication is the inadequacy of most flow cytometry analysis programs for the task; their primary purpose is to acquire and analyze data, placing little emphasis on producing a well-designed graphic for the purposes of meeting posters, publications, slides, grants, etc. Fortunately, images from most flow cytometry programs can be saved and exported to one of several powerful graphics programs for arrangement into figures. The Flow Core currently has both Macintosh and Pentium PC computers for this purpose, loaded with the software necessary to transform raw flow data into graphics suitable for publications. For the PC we have Adobe PhotoShop and Corel Photo-Paint, which can be used to make image edits (removal of unwanted labels, addition of legends, etc.) to flow images saved from CellQuest, WinMDI, etc. For production of the final figure we have Microsoft PowerPoint and Corel Draw, which can be used to prepare montages of flow histograms or cytograms, or integrate flow data with text, charts, graphs, etc. Most of our Macintosh computers have Canvas for image editing and PowerPoint for arranging figures. The Flow Core can provide individual assistance and training in the use of these programs to prepare figures, and is willing to set up workshops for interested groups.

How you can contribute to the upcoming Purdue University Cytometry Laboratory Cytometry CD-ROM (volume 3). Dr. J. Paul Robinson and his colleagues at the Purdue University Cytometry Laboratory have for several years published a series of CD-ROMs devoted to providing flow cytometry resources to the research community (including analysis programs, images, methods, educational materials, etc.). These CD-ROMs (currently in two volumes) have provided an invaluable information resource for both the experienced flow cytometry users and for investigators just getting into the field. Dr. Robinson’s group is currently assembling the material for a third volume; this edition will be primarily devoted to clinical applications of flow and will emphasize the detection of apoptosis and analysis of DNA. For this most recent edition the HSS Flow Core has been asked to serve as Section Editor for the DNA Analysis category. If your laboratory has a good protocol for DNA analysis (particularly for a specialized type of normal or malignant tissue), we would like to include it on the Purdue CD-ROM. While there are no financial rewards here (this is strictly a non-profit operation), you would receive the satisfaction of giving hundreds of laboratories access to your methodology through this unique resource (your submission can be cited in the literature by people who use it, too). Let me know if you would like to contribute. We also have both previous volumes if you would like to see what they are like. The deadline for submission is the end of August.

That’s it for now. Watch for our upcoming web page (by the end of summer, I hope!).

Cheers!

Bill

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