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What is it?

The LSR II (BD Biosciences) is a four-laser, sixteen-color cell analyzer.

What can it do

The LSR II has four lasers, 405, 488, 552, and 637 nm, and can detect light in seventeen photomultiplier tubes (PMTs) and one photodiode. Sixteen PMTs detect fluorochromes, one PMT detects side scatter, and the photodiode detects forward scatter.  The LSR II’s primary function is to characterize complex populations of cells. The LSR II can collect data at a rate up to 35,000 events per second, although it is typically used at much lower speeds. We have significantly improved the functionality of the BD LSR II in recent weeks. We added a 552 nm laser and expanded our ability to detect fluorochromes excited by the 405 nm laser. In addition, the 405 nm laser has been upgraded from a 25 mW gas-charged model to a 100 mW solid-state laser, which will generate a better signal, particularly from cells that weakly express your marker.

As technology allowed the field of flow cytometry to expand, the number of available fluorochromes and fluorescent proteins has greatly increased. For example, the discovery of Green Fluorescence Protein (GFP) earned Osamu Shimomura, Martin Chalfie, and Roger Y. Tsien the 2008 Nobel Prize in Chemistry. Since the discovery of GFP, numerous mutations have been introduced to the fluorescent protein that alter its emission and excitation spectra. For example, Clontech Laboratories, Inc (Mountain View, CA) currently has sixteen fluorescent proteins available; twelve of these proteins are most optimally excited by 552 nm lasers. Other companies sell dozens of additional variants of GFP.

In addition to exciting a variety of fluorescent proteins, the 552 nm laser allows us to optimize fluorochromes currently used, particularly phycoerythrin (PE) and PE-conjugated tandem dyes. PE can only be excited with 61% efficiency by a 488 nm laser, but this increases to 88% efficiency when stimulated with a 552 nm laser. This enhanced efficiency will enable users to visualize dimly stained cells. In addition, users may find that they are able to reduce the amount of antibody required for their assays, thereby reducing costs. Further, the new configuration of the instrument will allow the use of PE-Texas Red and the simultaneous use of PE-Cy5 from PE-Cy5.5.

The other major upgrade was the addition of detectors to the 405 nm laser. Previously, we could detect two fluorochromes excited by the 405 nm laser. One PMT detected light emitted from fluorochromes such as DAPI, Live/Dead Violet, Pacific Blue, or Horizon V450. The other PMT detected light emitted from fluorochromes such as Pacific Orange or Horizon V500. Major antibody manufacturers, such as BD Bioscience and eBioscience, have recently developed a series of dyes that are excited by the 405 nm laser, but emit light of a variety of wavelengths. For this reason, we added four PMTs to the 405 nm laser so that users can still use the fluorochromes to which they are accustomed, but also expand their ability to use the new technologies.

How does it work?

Individual cells pass single file through a stream. The stream is interrogated by four lasers and the light that is emitted from the fluorochromes is channeled into photomultiplier tubes (PMTs) that detect the light. The instrument’s electronics calculate the length of time required for a cell to pass through each laser intercept and assigns the signal from the PMT to the appropriate cell. In addition to measuring the fluorescent properties of each cell, physical properties are also measured. One photomultiplier tube detects Side Scatter (SSC), the light coming off cells at 90°. Side Scatter is a measure of the internal complexity of a cell; some refer to this parameter as “granularity”. The photodiode measures Forward Scatter (FSC), the light scattered at 0-8°. Forward Scatter is a rough measure of cell size.  Lymphocytes are small homogenous cells, so the FSC and SSC values are both small. Macrophages are large granular cells, so the FSC and SSC values are both large.

diagram of how flow cytometry worksFigure 1. How flow rate is changed. Low flow rate generates a more focused stream and higher resolution. High flow rate broadens the stream, resulting in lower resolution.

Sample flow rate control is set by the three flow rate control buttons: low, medium, and high. The approximate flow rates for these settings are 12, 35 and 60 µl/minute, respectively. The Sample Fine Adjustment knob allows the user to modify these settings from 0.5-2X. The instrument increases the sample rate by widening the core stream (Fig. 1).

The LSR II has precisely engineered light pathways. The fluorescent light emitted by your cells is channeled to the proper PMT by optical fibers, mirrors and filters.  For example, if your cells are stained with the fluorochrome FITC, FITC will be excited by the 488 nm blue laser and emit photons that are directed through the optical fibers of the 488 nm laser.

FAQs about the LSR II?

Do I really need to titer my antibodies?

Absolutely. Titering antibodies and other fluorochromes is essential to optimize the quality of your data. Using too much of one reagent could cause difficulty in getting the signal “on scale” and could cause difficulty with compensation of the other fluorochromes. In addition, using too much of a reagent costs money. Using too little of a fluorochrome could make your signal difficult to detect. A little investment before the experiment can pay enormous dividends later in the form of higher quality data and lower reagent costs.

What is the difference between Height, Area, and Width?

diagram of how flow rate is changedFigure 2. How data is acquired by flow cytometry. As the cells traverse the laser, a signal is generated that results in a histogram measuring the signal intensity over time. The height is the maximum signal. The Area is the area under the curve. The Width is Area divided by Height.

The LSR II generates three parameters for each cell in each channel.  These values are Height, Area, and Width.  These parameters reflect the fact that the instrument measures signal intensity versus time for each cell. As the cell passes through the laser, a histogram of signal intensity is generated. The Height is the maximum value attained when the middle of the cell passes through the beam (Fig. 2). Area is the area under the curve for each cell. Width equals Area divided by Height.

How do I do doublet discrimination?

Doublet Discrimination excludes two cells passing through the laser intercept concurrently. Doublets have double the area and width values of single cells while their height values are roughly the same as single cells.

What software can I use to analyze my data?

Data from the LSR II may be analyzed using many commercially available software. In the FCCL, users have access to FACSDiva (BD Biosciences) and FlowJo (TreeStar, Inc). Users are also welcome to purchase individual licenses for these or other programs for use in their laboratories.

What happens to my data after it is collected?

Users are responsible for their own data. We have copies of all raw data collected by the LSR II since 2005 and our intention is to continue storing all data acquired on the LSR II. However, if we were to experience a catastrophic computer failure resulting in the loss of the data, we are not responsible for the data. There are no back-up data outside of the core. Be sure to take a copy of your data with you.

Flow Cytometry Core Laboratory

University of Kansas Medical Center
Flow Cytometry Core Laboratory
Mail Stop 3050
3037 Hemenway
3901 Rainbow Boulevard
Kansas City, KS 66160