Flow Core Newsletter

May-June 1997

This is what’s new in the HSS Flow Core...

The Becton-Dickinson FACSCalibur is up and running. Our new B-D FACSCalibur has been installed and is available for use. 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). Fluorochromes that can be used as a fourth color include allophycocyanin (APC), which has an excitation maxima of about 620 nm and an emission maxima of about 670 nm. This fluorochrome can be conjugated to proteins in same manner as phycoerythrin and is available commercially conjugated to an increasing number of mouse and human antibodies, as well as avidin. Cy5 is another fluorochrome that can be excited by a diode laser and detected by the new instrument; it can also be conjugated to antibodies for immunophenotyping and has an excitation/emission spectra similar to APC. It is not quite as bright as APC but is cheaper, is less susceptible to photobleaching and is easier to conjugate to proteins. It is also useful for applications where a low molecular weight fluorochrome is more appropriate.

With the FACSCalibur, four cell surface markers can be detected simultaneously, or three markers plus DNA content. Some typical fluorochrome combinations include:

For four cell surface markers:

For three surface markers plus DNA content:

The FACSCalibur will also be useful for measuring transmembrane potential, intracellular pH, glutathione levels, etc. with simultaneous detection of three surface markers.

In addition, the FACSCalibur is equipped with a closed sorting system. Unlike the Vantage (which separates cells by breaking the sample stream into droplets and deflecting them with charged electrostatic plates) the Caliber sorts cells by means of a mechanical "catcher", which moves in and out of the sample stream to capture the wanted cell type. Since this system is completely enclosed, it can be used to sort biohazardous samples.

Call us to arrange instrument training and for help with designing experiments. Except for the second laser, the FACSCalibur will operate much like the FACScan.

A note to users of PE-Cy5 tandem dye conjugates. Investigators using PE-Cy5 tandem dye conjugates (sold under the trade names CyChrome, TriColor, Quantum Red, Red670, etc.) in the FL3 far red detector of the FACScan may find their use difficult when analyzing four colors on the FACSCalibur. These dyes are a conjugate of phycoerythrin and the cyanin dye Cy5. When cells labeled with this tandem conjugate pass through the diode laser, the Cy5 portion of the conjugate is excited; the resulting Cy5 emission will likely interfere with the signals from an APC or Cy5-conjugated antibody also on the surface of the cell. Use of PE-Cy5 simultaneously with APC may be possible but will require a great deal of compensation, which will lower sensitivity. An alternative is to use the B-D fluorochrome PerCP instead of PE-Cy5; this dye is excited by the argon laser and emits in the same range as PE-Cy5, but is not excited by the diode laser. Of course, you can still do three-color analysis on the FACSCaliber with PE-Cy5 conjugates as you have done previously on the FACScan, with the diode laser off.

APC, Cy5 and PerCP-conjugated secondary reagents available for use. To introduce users to the four-color capabilities of the FACSCalibur, the Flow Core has purchased small quantities of APC, Cy5 and PerCP-conjugated secondary labeling reagents. These include:

These secondary reagents can be used with the primary and biotin-conjugated antibodies already in use in your labs; thus, you can try expanding your detection repertoire without a major expenditure in direct conjugates. Contact Bill if you are interested in trying any of these reagents. These reagents are meant for pilot experiment use only; if you plan to make extended use of any of these conjugates, please plan to buy your own after the first two or three tries.

Preparation of Cy5-conjugated antibodies. We can help you prepare Cy5 conjugates of your primary antibodies and other proteins. The APC conjugation chemistry is still over my head (although Andy Beavis might be able to help with this if you are interested!).

Vendors for APC- and Cy5-conjugate antibodies. For both primary and secondary antibody conjugated to APC, try Pharmingen and Caltag; their lists of APC conjugates are constantly growing. Becton-Dickinson is the sole supplier of PerCP-conjugated avidin, secondary antibodies and a limited number of primary antibodies; their list is also is growing.

The B-D Vantage is fully operational. The Vantage cell sorter is running again and has been used successfully several times for sorting both mouse and human cells. The Vantage can also be used for three, four- or five-color experiments. Its UV laser makes it able to excite the Hoechst dyes (available in the Core) for DNA analysis and detection of apoptosis, and indo-1 for calcium flux measurement. We can also help you conjugate your antibodies to AMCA, a UV-excitable fluorochrome that emits in the blue at about 430 nm. The Hoechst dyes or AMCA can be used simultaneously with FITC, PE, PE-Cy5 and PerCP for up to five-color simultaneous analysis, if desired!

New version of CellQuest available. The latest updates of CellQuest (ver. 3.1) and ModFit (ver. 2.0) have been installed on all of the workstations. The new CellQuest version has an improved Attractors function (for automatic calculation of gating regions) as well as improved features for batch analysis of flow files. In addition, the Ground floor workstation in the Research Building will now successfully run both CellQuest and ModFit (the missing security module has been replaced).

Network access by the Mac workstations. We’re working on it! I’m told the problem is not with our computers but with the network, which currently does not have the software necessary to talk to Macs. It’s on Steve Taylor’s list of things to do. Hopefully by the time you read this it will be fixed. AppleTalk on the Macs themselves is up again (and includes the FACSCalibur and its printer), so you can network within the Core computers

Scheduling and record keeping in the Flow Core. We now have two sign-up sheets, one for the FACScan and one for the FACSCalibur. Remember to put down your phone number when you sign up, in case we have to contact you (about instrument malfunctions, etc.). We have also revised the user logs to make more room for descriptions of what users are running on the flow cytometers. Please take a few minutes and fill in the number and description of fluorochromes you are using, as well as what antibodies they are conjugated to (or if you are using PKH26, FITC-annexin V, DNA dyes, GFP, etc., etc.). This information will be necessary for the competitive renewal of our Core grant this fall; we need to provide descriptions of what sorts of new applications investigators are using the instruments for.

That's it. Cheers!

Bill

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