BD LSRII Operation instructions

BD Biosciences
2350 Qume Drive
San Jose, CA
95131-1807
Draft Rev A
September 2002
BD LSRII Overview
Workbook

ii BD LSRII Overview Workbook Draft

iii Draft Copyright
BD Biosciences • 2350 Qume Drive • San Jose, California
• 95131-1807
© 2002 Becton, Dickinson and Company. All rights reserved. No part of
this publication may be reproduced, transmitted, transcribed, stored in
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BD Biosciences, 2350 Qume Drive, San Jose, CA 95131, United States of
America.
BD Biosciences reserves the right to change its products and services at
any time to incorporate the latest technological developments. Although
this guide has been prepared with every precaution to ensure accuracy,
BD Biosciences assumes no liability for any errors or omissions, nor for
any damages resulting from the application or use of this information.
This workbook is subject to change without notice. BD Biosciences
welcomes customer input on corrections and suggestions for
improvement.
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For Research Use Only. Not for use in diagnostic or therapeutic
procedures.

iv BD LSRII Overview Workbook Draft

v Draft Table of Contents
• • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •
Table of Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-1
About the Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-3
Power Switch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-3
Control Panel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-4
Fluidics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-4
Optics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-9
Electronics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-14
Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-14
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-15
2 Operations and Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-1
Lab Exercise: BD LSRII Startup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-3
Lab Exercise: BD LSRII Shutdown . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-7
BD LSRII Maintenance and Care Procedures . . . . . . . . . . . . . . . . . . . . . . . . 2-9
Regular Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-9
Periodic Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-13
Laser Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-18
3 Optics and Lasers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-1
Lasers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-3
Optical Filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-4
Optical Filters in the BD LSRII . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-4
4 Instrument Quality Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-1
Instrument Quality Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-3
Lab Exercise: Performing Quality Control . . . . . . . . . . . . . . . . . . . . . . . . . . 4-4
Preparing the QC Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-4
Appendix A Worksheets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-1
Appendix B Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-1
BD LSR Overview Course Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-1

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Draft
1
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•
Introduction
After completing this module, you will be able to:
◆
describe the three systems of a flow cytometer
◆
describe the anatomy BD LSRII flow cytometer

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About the Instrument
The BD LSRII is a ten-color benchtop flow cytometer. It combines ease of
use with versatility and performance for advanced research applications.
Figure 1-a BD LSRII flow cytometer with outer covering removed
Power Switch
The power switch is located on the lower-right side of the BD LSRII
instrument.

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Control Panel
The fluidics controls, which consists of six buttons and one knob, are
found on the control panel in the front of the instrument.
Figure 1-b BD LSRII Control Panel
Fluidics
The purpose of the fluidics system is to carry the sample out of the sample
tube and into the sensing region of the flow cell. Cells are carried in the
sample core stream in single file and measured individually.
The fluidics system in the BD LSRII flow cytometer is pressure driven, a
built-in air pump provides a sheath pressure of 6.0 psi. After passing
through the sheath filter, sheath fluid is introduced into the lower chamber
of the quartz flow cell.
The sample to be analyzed arrives in a separate pressurized stream. When
a sample tube is placed on the sample injection port (SIP), the sample is
forced up and injected into the lower chamber of the flow cell by a slight
overpressure relative to the sheath fluid. The conical shape of the lower
chamber creates a laminar sheath flow that carries the sample core
upward through the center of the flow cell, where the particles to be

1-5Draft Module 1: Introduction
measured are intercepted by the laser beam. This process is known as
hydrodynamic focusing.
Figure 1-c BD LSRII flow cell
The objective in flow cytometric analysis is to have one cell or particle
moving through the laser beam at a given moment. The difference in
pressure between the sample stream and sheath fluid stream can be used
to vary the diameter of the sample core. Increasing the sample pressure
increases the core diameter and therefore the flow rate.
•A higher flow rate is generally used for qualitative measurements such
as immunophenotyping. The data is less resolved but is acquired more
quickly.
•A lower flow rate is generally used in applications where greater
resolution is critical, such as DNA analysis.
Proper operation of fluidic components is critical for particles to intercept
the laser beam properly. Always ensure that the fluidics system is free of
air bubbles and debris and is properly pressurized.
Flow Rate Control
Three flow rate control buttons—LO, MED, and HI—set the sample flow
rate through the flow cell. The sample fine adjust knob allows you to
adjust the rate to intermediate levels.
When the sample fine adjust knob is at its midpoint, the sample flow rates
at the LO, MED, and HI settings are approximately 12, 35, and 60µL/min
of sample, respectively. The knob turns five full revolutions in either
direction from its midpoint, providing sample flow rates from 0.5–2X the

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midpoint value. For example, if the LO button is pressed, the knob will
give flow rates from approximately 6–24 µL/min.
Fluid Control
Three fluid control buttons—RUN, STNDBY, and PRIME—set the
instrument operation mode:
• RUN pressurizes the sample tube to transport the sample through the
sample injection tube and into the flow cell. The RUN button is green
when the sample tube is on and the support arm is centered. When the
tube support arm is moved left or right to remove a sample tube, the
instrument switches to an automatic standby status to conserve sheath
fluid—the RUN button changes to orange.
•STNDBY (standby) restricts fluid flow to conserve sheath fluid. When
leaving the instrument for more than a few minutes, place a tube
containing 1 mL of deionized water on the sample injection port (SIP)
and press STNDBY.
• PRIME prepares the fluidics to begin a run by draining and filling the
flow cell with sheath fluid. The fluid flow initially stops and pressure is
reversed to force fluid out of the flow cell and into the waste container.
After a preset time, the flow cell fills with sheath fluid at a controlled
rate to prevent bubble formation or entrapment. At completion, the
instrument switches to STNDBY mode.
Sample Injection Port
The sample injection port (SIP) is where the sample tube is installed. The
SIP includes the sample injection tube and the tube support arm. Samples
are introduced through a stainless steel injection tube equipped with an
outer droplet containment sleeve. The sleeve works in conjunction with a

1-7Draft Module 1: Introduction
vacuum pump to eliminate droplet formation of sheath fluid as it
backflushes from the sample injection tube.
Figure 1-d BD LSRII Sample injection port
■Sample injection port (SIP)
• Sample injection tube—stainless steel tube that carries sample
from the sample tube to the flow cell. This tube is covered with
an outer sleeve that serves as part of the droplet containment
system.
• Tube support arm—arm that supports the sample tube and
activates the droplet containment system vacuum. The vacuum
is on when the arm is positioned to the side and off when the
arm is centered.
• Droplet containment system—prevents sheath fluid from
dripping from the SIP and provides biohazard protection.
When no sample tube is installed on the SIP, sheath fluid
backflushes through the SIP. This backflush helps prevent
carryover of cells between samples. The droplet containment
system vacuum is activated when the sample tube is removed
and the tube support arm is moved to the side. Sheath fluid is
aspirated as it backflushes the sample injection tube.
mCAUTION: If a sample tube is left on the SIP with the tube support arm to
the side (vacuum on), the sample will be aspirated into the waste container.

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Sheath and Waste Tanks
The sheath and waste tanks are outside the instrument. They should be
kept in the same location the field service engineer placed them at
installation. Moving the tanks to a different location, i.e. from the floor to
the benchtop, will change the sheath velocity.
The sheath tank is a metal container with a capacity of 8 L. Sheath fluid is
filtered through an in-line, interchangeable filter that prevents small
particles from entering the sheath fluid lines. The sheath filter is located on
top of the sheath tank.
Before opening the sheath tank:
1 Put the instrument in STNDBY mode.
2 Disconnect the air line (green).
3 Depressurize the sheath tank by lifting its vent cap.
The waste tank is plastic container with a capacity of 10 L. The waste
tank is equipped with a waste management system that consists of a base
and an overfull sensor. The base holds the waste tank and is equipped
with magnets to hold the base to the leg of a metal bench or table to
prevent tipping. The overfull sensor will emit an audible alarm when the
waste is full. To test the alarm, press the test button on the base.
HWARNING: To avoid leakage of biohazardous waste, put the instrument
in STNDBY mode before disconnecting the waste container.
HWARNING: The waste container contents might be biohazardous. Treat
contents with bleach (10% of total volume). Dispose of waste with proper
precautions in accordance with local regulations. Wear suitable protective
clothing, eyewear, and gloves.
Recommended Sheath Fluids
• BD FACS Flow™ sheath fluid (BD Biosciences, Catalog no. 342003)
• Phosphate-buffered saline (PBS) (Dulbecco’s Ca++-free and Mg++-
free) for certain applications

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Non -Recommended Sheath Fluids
• Fisher Hematology Diluent
• Isoton III
• Isolac D
• Deionized water
NOTE: If you make your own sheath fluid in the lab, be sure to pass it
through a 0.22-um filter before running it on the BD LSRII instrument.
Optics
The optics consists of:
• lasers that generate excitation light
• filters and mirror that route the laser light to the fluidic stream
• fiber optic cables that direct the resulting light scatter and fluorescence
signals to the appropriate emission block
• filters that direct the light scatter and fluorescence signals in the
emission block to the appropriate PMT
Light Scatter
When a cell or particle passes through a focused laser beam, laser light is
scattered in all directions. Light that scatters axial to the laser beam is
called forward scatter (FSC); light that scatters perpendicular to the laser
beam is called side scatter (SSC). FSC and SSC are related to certain
physical properties of cells:
• FSC—indicates relative differences in the size of the cells or particles
• SSC—indicates relative differences in the internal complexity or
granularity

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Fluorescence
When cells or particles stained with fluorochrome-conjugated antibodies
or other dyes pass through a laser beam, the dyes can absorb photons
(energy) and be promoted to an excited electronic state. In returning to
their ground state, they release energy, most of which is emitted as light.
This light emission is known as fluorescence.
Fluorescence is always a longer wavelength (lower-energy photon) than
the excitation wavelength. The difference between the excitation
wavelength and the emission wavelength is known as the Stokes shift.
Some fluorescent compounds such as PerCP exhibit a large Stokes shift,
absorbing blue light (488 nm) and emitting red light (675 nm), while
other fluorochromes such as FITC have a smaller Stokes shift, absorbing
blue light and emitting green light (530 nm).
Lasers
The basic BD LSRII flow cytometer has fixed alignment, 488-nm laser
excitation optics. Up to three additional lasers can be added. All of the
optional lasers are also alignment-free.
• The primary, Coherent Sapphire, solid state 488nm laser generates
forward scatter (FSC) and side scatter (SSC–PMT1) signals and up to
four fluorescence signals—, PMT2, PMT3, PMT4, and PMT5
• The optional VioFlame 405nm laser generates two fluorescence
signals—PMT6 and PMT7
• The optional Kimmon HeCd 325nm or Lightwave solid state 355nm
laser generates two fluorescence signals—PMT8 and PMT9
• The optional JDS Uniphase HeNe 633nm laser generates two
fluorescence signals—PMT10 and PMT11
mCAUTION: To extend the life of the HeCd laser, turn on the instrument for
at least 4 hours at least once per week.

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Table 1-1 BD LSRII Laser Summary
Filters
Optical filters attenuate light or help direct it to the appropriate detectors.
The BD LSRII instrument uses dichroic filters. Dichroic filters transmit
light of a specific wavelength, while reflecting other wavelengths. The
name and spectral characteristics of each filter appear on its holder.
There are three types of dichroic filters:
• Shortpass (SP) filters transmit wavelengths that are shorter than the
specified value.
• Longpass (LP) filters transmit wavelengths that are longer than the
specified value.
• Bandpass (BP) filters pass a narrow spectral band of light by
combining the characteristics of shortpass filters, longpass filters, and
absorbing layers. Discriminating filters (DF) are a type of bandpass
filter.
Bandpass and discriminating filters have the same general function—they
transmit a relatively narrow band of light. The principle difference
between them is their construction. Discriminating filters have more
cavities or layers of optical coatings, resulting in a steeper transmission
curve than the curve for a Bandpass filter. This steep slope means that a
Laser Type
Wavelength
(nm) Power
Warm-up
Time (min)
Coherent Sapphire 488 (blue) 20 mW 30 min.
Kimmon HeCd or
Lightwave solid state
325 or 355
(UV)
8mW or 20mW
(preliminary)
60 min.
Coherent VioFlame 405 (violet) 25 mW 15 min.
JDS Uniphase 1344 P633 (red) 17 mW 20 min.

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discriminating filter is better at blocking light outside the rated bandwidth
of the filter.
Figure 1-e Optical Filters

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When dichroic filters are used as steering optics to direct different color
light signals to different detectors, they are called dichroic mirrors or
beam splitters.
• Shortpass dichroic mirrors transmit shorter wavelengths of light to
one detector while reflecting longer wavelengths to a different
detector.
• Longpass dichroic mirrors transmit longer wavelengths to one
detector while reflecting shorter wavelengths to a different detector.
The default optical configuration on the BD LSRII uses all longpass
dichroic mirrors to direct the light to the appropriate PMT.
Detectors
Light signals are generated as particles pass through the laser beam in a
fluid stream. When these optical signals (photons) reach a detector they
are converted to electrical pulses. The electrical pulses then are digitized
into one of 16,384 possible levels 10 millions times per second by analog-
to-digital converters. The digital signal is then further processed by the
electronics system.
There are two types of signal detectors in the BD LSRII flow cytometer:
the photodiode and photomultiplier tubes (PMTs). The photodiode is less
sensitive to light signals than the PMTs, thus is used to detect the stronger
FSC signal. PMTs are used to detect the weaker signals generated by SSC
and all fluorescence channels. These signals are amplified by applying a
voltage to the PMTs. As the voltage is increased, the detector sensitivity
increases, resulting in increased signal. As the voltage is decreased, the
detector sensitivity decreases, resulting in decreased signal. Detector
voltages are adjusted in the Instrument Settings Inspector in the DigFACS
software, as described on page 71 of the FACSDiva User’s Guide.
Emission Blocks
Emission blocks house the PMTs and the dichroic and bandpass filters
used to direct emitted light. There are two types of emission blocks in the
BD LSRII flow cytometer: the octagon and trigon blocks. The octagon
emission block is used for light excited by the 488nm laser. An octagon
can house up to seven different PMTs, measuring up to 6 fluorescent
channels and side scatter light. The trigon emission blocks are used for

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light excited by the UV, 405nm, and 633nm lasers. Each trigon can house
up to two PMTs, measuring two fluorescent channels.
Electronics
The digital electronics found in the BD LSRII digitizes the amount of light
signals created by particles passing through the laser beams. The voltage
corresponding to each signal is digitized continuously during operation
and is represented as numbers in the computer’s memory.
Threshold
The threshold defines the level at which the system starts to look for
pulses. The system continuously monitors the data and simultaneously
calculates area and height for all channels each time a signal exceeds the
threshold.
Software
The BD DigiFACS installer installs the following applications:
• BD DigiFACS software, for acquiring and analyzing data
• Data Manager utility, for backing up, archiving, and restoring data
•Sentinel System Driver, needed to use the security module see: Chapter
2: BD FACSDiVa Software Setup 31in the FACSDiva User’s Guide
• Java™ 2 Runtime Environment (JRE), needed to run BD DigiFACS
software
• Sybase™ SQL Anywhere™ Studio, needed to run the database
• Adobe® Acrobat® Reader, needed to view the PDF version of the
user’s guide