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LSR II

The Becton-Dickinson LSR II is a 5-laser 15-color analytical flow cytometer.

Training

Unless you are already conversant with the LSR II and FACSDiva software, you will need extra training before you can use the machine. To arrange training, proceed as outlined below:
  1. Familiarize yourself with the material covered in the You need not review all the material in this course, but please complete at least the following:
    • Introduction
    • Software Overview
    • CS&T Overview (skim content)
    • BD LSR II
    • Data Analysis
    • Data Management
    • Templates
  2. Once you have completed the on-line course, please print and read the following document
  3. Finally, Contact us to arrange training.
    Remember to bring the LSRII FACSDiva Quick Reference Guide to your training session.
    The initial training session is probably best done without cells - we have beads to use.
    Please do not plan important experiments for at least your first two sessions on the instrument.
You may also find the following material useful:
 

Using the LSR II

General

  • Buffer: Your cells should be suspended in PBS or phenol red-free HBSS or MEM. EDTA (~ 2 mM) may help minimize clumping. DNAse (~100 U/ml or 1 µg/ml) also helps. You may include up to 0.5% protein (i.e. 0.5% BSA, or 1% FBS). If using FBS, ensure that it is heat inactivated and dialyzed vs. calcium/magnesium-free PBS.
  • Volume: Resuspend in around 500 µl.
  • Concentration: Ideally cells should be at 1 x 106/ml. 2 x 105/ml should be considered a minimum usable concentration.
  • Tubes: Use Falcon 2052 (12 x 75 mm polystyrene) tubes. DO NOT USE ANY OTHER BRAND! Other brands may damage the cytometer seals. It is perfectly OK to bring cells in microcentrifuge tubes; we provide Falcon 12 x 75 mm tubes and you can transfer to these tubes when you run the cells.

Controls

In order to be able to interpret your data, you must bring the appropriate controls!
 
Single color.
For a simple single-color experiment you need only a negative and positive control. For single color fluorescent protein experiments determining transfection efficiency, you may not need anything other than your dilution series.
Multiple colors.
For multicolor experiments, the requirements are much more stringent because of the need to do color spillover compensation.
 
Controls for multicolor experiments.
 
Color compensation controls.
At mimimum, you must have all of your single-color controls, and a negative. Without these you will not be able to set up color compensation, and your data will be uninterpretable. The exceptions to this are (a) if you're using PI or DAPI to exclude dead cells, with a GFP or other green fluor, or (b) if you're using only Indo-1 or CCF2 or similar dyes.
 
You may find it better to use antibody capture beads, rather than cells, to set up compensation controls. Beads have the advantage that they are uniformly positive, and circumvent problems that might arise if your cells are weakly stained or the positive population is very small. At least BD, Invitrogen, and Spherotech sell capture beads.
 
FMO controls.
To properly interpret multicolor immunophenotyping experiments, you should also prepare FMO (Fluorescence Minus One) background controls. A FMO control is stained with every fluor except one, and is used to determine the post-compensation background for that fluor. If your cells stain brightly positive, FMO controls may not be needed; however they are very useful for dimly stained cells.