Evaluation of the Becton-Dickinson LSR.
We recently evaluated the Becton-Dickinson LSR, the latest benchtop flow cytometer developed by Becton Dickinson Biosciences. This instrument is equipped with an argon-ion laser like that found on the FACSCalibur and a helium-cadmium (HeCad) laser that emits in the UV range. The addition of this laser allows the use UV-excited fluorochromes (including the Hoechst dyes and DAPI, indo-1 and AMCA) in a benchtop sorter format, both alone and in conjunction with other fluorochromes. At present UV fluorochromes require a complex cell sorter with operator assistance; the LSR laser and detector alignment is "locked down" like the FACSCalibur, allowing the trained user to use these fluorochromes in a user-friendly format. The instrument uses a Vantage-type card cage (including full pulse-processing capability), a FACSCalibur-style flow cell and Vantage-style PMT detectors. All dichroics and bandpass filters are replaceable by the user, allowing great flexibility in experiment design. All three laser beams are spatially separated, allowing them to be used simultaneously. Time delays between the lasers is configured during installation and supposedly does not require user adjustment, but can be verified using FACSComp as on the FACSCalibur. The instrument does not allow crossbeam compensation between lasers as is found on the FACSVantage SE; all interlaser compensation manipulations need to be done post-experiment through software packages such as Verity WinList or FlowJo. This instrument should find applications in complicated experiments that previously required a more complex multilaser cell sorter.
The B-D LSR. The LSR with the cover off (below, left), and looking down the laser bay (below, right). The instrument is equipped with an air-cooled argon-ion laser emitting at 488 nm (20 mW) and a Kimmon air-cooled HeCad laser emitting at 325 nm (8 mW). A third HeNe laser emitting at 632 nm (17 mW) is also shown mounted on the platform and will be available for analysis in the near future. The PMT cluster is at the bottom of the right photo.
PMT and filter configuration of the B-D LSR. Photograph (below, left) and schematic diagram (below, right) of the LSR optical bench, showing the default dichroic/filter configuration. FL1, FL2, FL3 and FL6 (P3, P4, P7 and P8) receive fluorescent signals from the primary argon-ion 488 nm laser; FL4 and FL5 (P5 and P6) receive signals from the HeCad 325 nm laser (much as on the FACSVantage SE). A 620 nm shortpass dichroic separates the FL3 and FL6 signals (presumably red) and a subsequent 510 longpass dichroic separates the blue and near-UV signals originating from the HeCad laser. The remaining signals intended for FL1 and FL2 (usually FITC and PE) are split with a 555 longpass dichroic. Narrow bandpass filters are the same wavelength and tolerances as found on the FACSVantage SE. With the addition of a third HeNe laser, one of the red-detecting PMTs presently accepting signals from the argon-ion laser will be dedicated to HeNe-excited fluorochromes.
The following applications were evaluated...
Four-color immunophenotyping off the primary laser. The LSR optical configuration permits four-color analysis off the primary argon-ion laser. In the experiment below, mouse EL4 cells were labeled with biotin-anti-CD90 (Thy1.2) followed by secondary labeling with either FITC-, PE-, PE-Texas Red or PE-Cy5 avidin. A mixture of the individually labeled cells and unlabeled controls was then analyzed (top panel). The optical bench configuration for four color analysis is shown below (bottom panel). Four-color analysis was possible using roughly the same color compensation values as for similar analysis on the FACSVantage SE.
UV-stimulated DNA binding dyes. Hoechst 33342, Hoechst 33258 and DAPI are UV-excited DNA binding dyes that give particularly good stoichiometric binding. DAPI is particularly useful for very accurate DNA cell cycle analysis. Moreover, Hoechst 33342 is cell-permeant, allowing DNA content analysis in viable cells. The LSR with its quartz flow cell should be particularly useful for Hoechst dye and DAPI cell cycle analysis. This was found to be the case both with chick erythrocyte and calf thymocyte nuclei internal standards, and with cell lines (shown below).
(Below). Chick erythrocytes (top) and calf thymocyte nuclei (bottom) labeled with DAPI at 2 µg/ml and analyzed on the LSR (left panels). C.V.s were excellent for both cell standards, and the detector linearity was acceptable based on the ratios between the mean fluorescence intensities of the chick erythrocyte singlet (peak 1), doublet (peak 2) and triplet (peak 3) populations. For DAPI and Hoechst dye analysis, a 424/44 bandpass filter was used in front of the FL5 PMT (right). Data courtesy of Dr. Gerald Marti (FDA).
(Below). EL4 cells were either fixed and labeled with DAPI or Hoechst 33342 at 2 µg/ml (left and middle panels), or incubated live with the cell-permeable Hoechst 33342 at 2 µg/ml for 30 minutes (right panels). Cells were then analyzed on both the FACSVantage SE and the LSR. Although both instruments gave acceptable results, the LSR with its quartz flow cell gave better G1 C.V.s than the FACSVantage SE, particularly in viable cell analysis. The FACSVantage SE was modified for this experiment with a 50 µm nozzle for maximum resolution.
UV-stimulated protein-conjugated fluorochromes. UV-excited hapten fluorochromes such as 7-aminomethylcoumarin (AMCA) have seen limited use in flow cytometry due to the strong cellular autofluorescence in the blue region of the spectrum. Nevertheless, they can sometimes be used to label very dense antigens when the appropriate UV laser is available, although they are generally dimmer than other hapten fluorochromes such as fluorescein (below, top panel; filled peak, specific labeling; unfilled peak, isotype matched control). In this experiment, EL4 cells were labeled with biotin-anti-CD90 followed by Alexa Fluor 350-conjugated avidin (a form of AMCA available from Molecular Probes). The LSR HeCad laser appeared to stimulate AMCA as well as a more powerful (100 mW) krypton-ion laser emitting at 351 nm on a FACSVantage SE (below, middle and lower panels). For this experiment, the 380 LP filter in front of the FL5 PMT was replaced with 424/44 narrow bandpass (below, right).
ELF-97 ™ phosphate alkaline phosphatase substrate. ELF-97™ phosphate (Molecular Probes, Inc.) is a fluorogenic phosphatase substrate. The cleaved alcohol form of ELF-97 has an excitation maxima in the UV and emits strongly in the same spectral range as fluorescein (around 530 nm), making it a very useful reagent for detecting both endogenous alkaline phosphatase and AP-conjugated reagents. In this experiment, EL4 cells were labeled with biotin-anti-CD90 (Thy1.2) as above and secondary labeled with AP-conjugated avidin, followed by ELF-97 labeling. ELF-97 fluorescence was detected by the inserting a 530/30 bandpass filter in front of the FL5 PMT in place of the default 380 LP (below, right panel). The left panel below shows the resulting Thy1.2 detection (blue peak) compared to an isotype control antibody with all subsequent labeling steps (unfilled peak). The HeCad 325 nm laser line proved to be as efficient as previously tested argon- and krypton-ion UV lines for exciting the ELF-97 alcohol. Click here for more information on the ELF-97 substrate for flow cytometry.
Indo-1 detection of calcium flux. The ratiometric nature of indo-1 makes it one of the most useful probes for measuring intracellular calcium flux. Indo-1 analysis has previously been restricted to cell sorters equipped with water-cooled argon- and krypton-ion lasers emitting at UV wavelengths. In this experiment, EL4 cells were labeled with indo-1 AM (Molecular Probes) at 5 µM for 30 minutes at 37oC, washed three times and analyzed simultaneously on the LSR and a FACSVantage SE. Ionomycin was added to the indo-1-labeled cells following 30 seconds of baseline fluorescence collection to trigger calcium influx. The FACSVantage was set up to detect indo-1 in the third laser position, using 395/10 and 495/10 filters for detecting bound and unbound indo-1 signals respectively, a 460 LP extended transmission filter to split the signals and krypton-ion 351 nm laser excitation at 100 mW (go here for more information about indo-1 on the Vantage). The LSR used the filter configuration shown below in the bottom panel. Both instruments showed comparable sensitivity as reflected in the bound/unbound indo-1 ratio (top panel), although the FACSVantage was slightly more sensitive in detecting the longer wavelength unbound indo-1 signal (probably a consequence of somewhat poorer excitation of this form by the HeCad 325 nm laser line).
LSR II. The current optical bench layout of the LSR is below. This configuration allows the analysis of 4 colors off the primary argon-ion laser, two colors off the UV and two off the HeNe (the 4-2-2 configuration).
More information about the LSR can be obtained from Becton-Dickinson Biosciences. David Coder's laboratory at the University of Washington is equipped with an LSR; his review of the instrument can be found here.