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BD LSRII Workbook

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

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  • Page 1 BD LSRII Overview Workbook BD Biosciences 2350 Qume Drive San Jose, CA 95131-1807 Draft Rev A September 2002...
  • Page 2 BD LSRII Overview Workbook Draft...
  • Page 3 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.
  • Page 4 BD LSRII Overview Workbook Draft...

  • Page 5: Table Of Contents

    Lab Exercise: BD LSRII Startup ........2-3...
  • Page 6 BD LSRII Overview Workbook Draft...

  • Page 7: 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 Draft...
  • Page 8 BD LSRII Overview Workbook Draft...

  • Page 9: About The Instrument

    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.

  • Page 10: Control Panel

    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...
  • Page 11 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...
  • Page 12 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 BD LSRII Overview Workbook Draft...
  • Page 13 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.
  • Page 14 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 BD LSRII Overview Workbook...

  • Page 15: Optics

    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 •...
  • Page 16 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.
  • Page 17 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.
  • Page 18 filter is better at blocking light outside the rated bandwidth of the filter. Figure 1-e Optical Filters 1-12 BD LSRII Overview Workbook Draft...
  • Page 19 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.

  • Page 20: Electronics

    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.

  • Page 21: Overview

    Overview Control Panel Briefly describe each of the fluid control buttons shown in Figure 1-f. Figure 1-f ________________________________________________________ ________________________________________________________ ________________________________________________________ STNDBY ________________________________________________________ ________________________________________________________ ________________________________________________________ PRIME ________________________________________________________ ________________________________________________________ ________________________________________________________ Draft Module 1: Introduction 1-15...
  • Page 22 What happens if the tube support arm is directly under the sample tube and the cytometer is in STNDBY? ________________________________________________________ ________________________________________________________ ________________________________________________________ What happens if the tube support arm is left to the side when a sample tube is on the SIP? ________________________________________________________ ________________________________________________________ ________________________________________________________ 1-16 BD LSRII Overview Workbook Draft...
  • Page 23 Sheath and Waste Tanks Where should the sheath and waste tanks be placed? ________________________________________________________ What is the capacity of the sheath tank and the waste tank? ________________________________________________________ The instrument must be left in ________________ before disconnecting the waste container. Draft Module 1: Introduction 1-17...
  • Page 24 1-18 BD LSRII Overview Workbook Draft...

  • Page 25: Operations And Maintenance

    Operations and Maintenance • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • After completing this module, you will be able to: Perform the startup and shutdown procedures Describe the regular maintenance and periodic maintenance procedures...
  • Page 26 BD LSRII Overview Workbook Draft...

  • Page 27: Lab Exercise: Bd Lsrii Startup

    Remove the lid of the sheath tank. Fill the tank with sheath fluid. Typically an isotonic saline solution is used as sheath fluid. BD FACSFlow solution (BD Biosciences Catalog No. 342003) is recommended and used as sheath fluid during the training course.
  • Page 28 Add a sufficient quantity of bleach to obtain a minimum 10% final concentration of bleach. Replace the waste tank lid and reconnect the orange line to the orange connector on the waste tank. Removing Air Bubbles First, bubbles will be removed from the fluidics lines. BD LSRII Overview Workbook Draft...
  • Page 29 Check the sheath filter for trapped air bubbles. If bubbles are visible, gently tap the filter body with your fingers to dislodge the bubbles and force them to the top. Firmly pinch the tubing between your fingers and loosen the sheath filter’s vent cap to bleed off any air in the filter.
  • Page 30 The STNDBY button lights yellow after priming is complete. Install a 12 x 75-mm tube with 1 mL of DI water on the SIP and place the support arm under the tube. BD LSRII Overview Workbook Draft...

  • Page 31: Lab Exercise: Bd Lsrii Shutdown

    It will take approximately 15 minutes to complete the shutdown procedure. Always clean the BD LSRII instrument before it is turned off at the end of the day. Proper cleaning will ensure that your instrument will function consistently. Cleaning prevents the sample injection tube from becoming clogged and removes dyes that could remain in the tubing, causing carryover.
  • Page 32 flow cell. CAUTION: Do not leave more than 1 mL of water on the SIP. When the BD LSRII flow cytometer is turned off or left in STNDBY mode, a small amount of fluid will drip back into the sample tube.

  • Page 33: Bd Lsrii Maintenance And Care Procedures

    BD LSRII Maintenance and Care Procedures The BD LSRII instrument is designed to require minimum maintenance. However, to preserve the reliability of the instrument you must regularly perform basic preventive maintenance procedures. WARNING: All biological specimens and materials coming into contact with them are considered biohazardous.
  • Page 34 Press the PRIME button on the fluidics control panel. When the STNDBY button lights orange, press the PRIME button again. Install a tube with 3 mL of a bleach solution. Use BD FACS Clean or a 1:10 dilution of bleach in DI water as the bleach solution. 2-10...
  • Page 35 5 minutes. Optional System Flush Procedure Perform this procedure as an alternative to the bi-monthly bleach flush. This procedure has an additional rinse step that uses BD FACS Rinse, a detergent-based solution. Remove the sheath filter. Refer to Changing the Sheath Filter on page 23 in this workbook for sheath filter removal instructions.
  • Page 36 STNDBY button lights orange, press the Prime button again. Install a tube with 3 mL of bleach solution. Use BD FACS Clean or a 1:10 dilution of bleach in DI water as the bleach solution. Press the RUN fluid control button and run on HI for 30 minutes.

  • Page 37: Periodic Maintenance

    WARNING: Follow good laboratory practices; wear protective clothing, eyewear, and gloves whenever cleaning the instrument or replacing parts. Use the information in the table below to determine when to perform periodic maintenance procedures. Table 2-2 BD LSRII Periodic maintenance procedures Procedure When Changing the sheath Perform every 3–6 months.
  • Page 38 filter from the sheath container. Figure 2-a Sheath Filter for the BD LSRII Wrap approximately two layers of Teflon® thread tape around the threads of the new filter to ensure a proper seal. Write the date on the new filter.
  • Page 39 Connect the quick-disconnect. Turn on the instrument to pressurize the sheath container. Loosen the filter’s vent cap to bleed off any air in the sheath filter. Carefully tap the filter assembly to dislodge any air trapped in the filter element. Loosen the filter’s vent cap again to bleed off any air in the sheath filter.
  • Page 40 Tighten the retainer just enough to hold it in place. Slide the outer sleeve over the sample injection tube and into the opening of the retainer. Continue tightening the retainer. 2-16 BD LSRII Overview Workbook Draft...
  • Page 41 Install a sample tube on the SIP to ensure that the outer sleeve has been properly installed. If the sleeve hits the bottom of the tube, loosen the retainer slightly and push the sleeve up as far as it will go. Retighten the retainer. Changing the Sample Tube O-Ring The sample tube O-ring (Catalog No.

  • Page 42: Laser Maintenance

    Frequent startup and shutdown might cause damage to the laser tube. TIPS • Do not shutdown the BD LSRII cytometer within 30 minutes after the latest startup. • After shutting the BD LSRII cytometer off, wait 30 minutes before restart.

  • Page 43: Optics And Lasers

    • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • After completing this module, you will be able to: List optical filters and lasers used in a BD LSRII Describe the Octagon and Trigon optical pathways...
  • Page 44 BD LSRII Overview Workbook Draft...

  • Page 45: Lasers

    17 mW ______ min. Do not shutdown the BD LSR cytometer within 30 minutes after the latest startup. After shutting the BD LSR cytometer off, wait 30 minutes before restart. The primary, argon-ion, laser generates forward scatter (FSC) and side scatter (SSC) signals and up to four fluorescence signals.

  • Page 46: Optical Filters

    Optical Filters in the BD LSRII Table 3-2 describes the optical filters used for various fluorochromes with the BD LSRII. The PMT# corresponds to the PMT on the emission block, the channel corresponds to the channel in the DigiFACS software.
  • Page 47 BD LSRII Optical Bench Information Table 3-2 Primary Laser Fluorochrome PMT# Channel Dichroic Filter Other Fluorochrome(s) 488 nm (blue) Side Scatter 488/10BP FITC 505DCLP 530/30DF EGFP 555DCLP 575/26DF Use 575/26 with 600LP dichroic for 5-colors (PMT#4 = PE-Texas Red) from 488. Use 585/42 with 555LP for 4-colors.
  • Page 48 Emission Blocks The BD LSRII comes equipped with 4 emission blocks, one for each laser. 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.
  • Page 49 Cascade yellow 555 DCLP 525/50 HO violet laser fiber optic Cascade blue 450/50 AF VioFlame 405nm (violet) laser FL6 and FL7 Figure 3-b Indo-1 blue 450 DCLP 530/30 DF UV laser fiber optic Indo-1 violet 405/20 DF HeCd 325nm (UV) laser FL8 and FL9 Figure 3-c Draft Module 3: Optics and Lasers...
  • Page 50 Default Instrument Configuration The table below is a listing of the default instrument configuration shown in the software for the BD LSRII. For more information regarding instrument configuration see page 94 of the FACS Diva User’s Guide. BD LSRII Instrument Configuration...
  • Page 51 Parameter Laser Channel EGFP EYFP PE-Cy5 PE-Cy5-5 PerCP ECFP ß-Lactamase blue Alexa 430 ECFP to EYFP FRET ß-Lactamase green Alexa 350 AMCA Hoechst 33342 DAPI Alexa 633 * The default Instrument Settings parameters are indicated by shading. Draft Module 3: Optics and Lasers...
  • Page 52 BD LSRII Optics Exercise Using Table 3-2 on page 3-5 enter the dichroic and bandpass filters you would use for the following fluorochromes. Optics exercise Table 3-4 Fluorochrome Excitation Emission Dichroic Bandpass EGFP 489nm 508nm 480nm 578nm PerCP 490nm 675nm...
  • Page 53 Changing Optical Filters Some applications require the changing of optical filters. For example, calcium-flux experiments with indo-1 use a different filter from that used in DNA experiments with DAPI or Hoechst 33342. Use the following procedure to change filters. Lift the right lid of the instrument. Remove the desired filter.
  • Page 54 3-12 BD LSRII Overview Workbook Draft...

  • Page 55: Instrument Quality Control

    • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • After completing this module, you will be able to: Perform daily quality control on your BD LSRII Draft...
  • Page 56 BD LSRII Overview Workbook Draft...

  • Page 57: Instrument Quality Control

    Keep track of means and CVs in a quality control (QC) log. QC data should be analyzed for trends over the past 30–60 runs. NOTE: NOTE: QC results are affected by laser and fluidics performance. BD Biosciences strongly recommends following the laser and fluidics maintenance procedures in the found in this workbook. Draft...

  • Page 58: Lab Exercise: Performing Quality Control

    The steps in this section show you how to set up an Experiment for instrument quality control. If you have already created a similar Experiment, you can reuse it by duplicating or importing the Experiment. See Using Experiments as Templates on page 64 in the FACSDiva User’s Guide. BD LSRII Overview Workbook Draft...
  • Page 59 CAUTION: For accurate data results, the Instrument Configuration dialog box must reflect the physical layout of the BD LSRII optical bench. If you need to add additional parameters or change the current configuration, see Instrument Configuration on page 94 in the...
  • Page 60 Inspector, and add the CV and mean for only the FITC fluorochrome. Set the number of integers (#) to 1 for the CVs and 0 for the means. Repeat selecting statistics for each plot and BD LSRII Overview Workbook Draft...
  • Page 61 fluorochrome. For more information, see Using the Statistics Inspector on page 135 in the FACSDiva User’s Guide. In the Acquisition Controls frame, set the Number to Record to 10,000 evt. and the Events to Display to 500 evt. NOTE: Tip Decreasing the number of displayed events will increase the data refresh rate.
  • Page 62 To display the Population Hierarchy view, right-click the plot and choose Show Population Hierarch. Select a population in the Hierarchy view and enter a new name to change it. For example, change P1 to FSC, P2 to SSC, and P3 to FITC. BD LSRII Overview Workbook Draft...
  • Page 63 Edit the statistics view to show only the named populations. Select the statistics view. Click on the Populations tab in the Statistics Inspector and delete the All Events population. Copy the results into the QC log and print the worksheet for your records.
  • Page 64 4-10 BD LSRII Overview Workbook Draft...

  • Page 65: Appendix A Worksheets

    Appendix A Worksheets • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • Draft...
  • Page 66 BD LSRII Overview Workbook Draft...
  • Page 67 Figure A-a Octagon Draft Appendix A: Worksheets...
  • Page 68 Figure A-b Trigon violet BD LSRII Overview Workbook Draft...
  • Page 69 Figure A-c Trigon red Draft Appendix A: Worksheets...
  • Page 70 Figure A-d Trigon UV BD LSRII Overview Workbook Draft...

  • Page 71: Appendix B Troubleshooting

    Appendix B Troubleshooting • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • Draft...
  • Page 72 BD LSRII Overview Workbook Draft...
  • Page 73 • details of recent instrument performance In the US, call (800) 448-BDIS (2347), Option 1. In Canada, call (800) 268-5430. In other countries, contact your local BD Biosciences representative. Refer to our website, http://www.bdfacs.com, for up-to-date contact information. Draft Appendix B: Troubleshooting...
  • Page 74 No keyboard response Bad keyboard connection Check keyboard connections to dongle and computer. Communication failure • Reseat the computer. between computer and • Reseat the CAT5 cable, located instrument in back of the flow cytometer. BD LSRII Overview Workbook Draft...
  • Page 75 60 minutes for the UV laser, 15 minutes for the VioFlame laser, and 20 minutes for the HeNe laser. Laser not functioning Contact BD Biosciences. No events in acquisition RUN not activated Press the RUN button. display and RUN button...
  • Page 76 20 minutes. If the event rate is still erratic, clean the sample injection tube. Contaminated sample Prepare the specimen again, making sure that the tube is clean. BD LSRII Overview Workbook Draft...
  • Page 77 Observation Possible Causes Recommended Solutions Scatter parameters Instrument settings Optimize the scatter parameters. appear distorted maladjusted Air bubble in sheath filter or Remove the air bubble. flow cell Flow cell dirty Perform the system flush procedure. Air leak at sheath container Ensure that the sheath container lid is tight and all connectors are secure.
  • Page 78 FACSFlow™ and use FACSFlow for sheath fluid. Laser not warmed up Wait 30 minutes for the Sapphire laser, 60 minutes for the UV laser, 15 minutes for the VioFlame laser, and 20 minutes for the HeNe laser. BD LSRII Overview Workbook Draft...
  • Page 79 Draft Appendix B: Troubleshooting...
  • Page 80 B-10 BD LSRII Overview Workbook Draft...

  • Page 81: Bd Lsr Overview Course Evaluation

    Training Date: ____________________________________________________________ Instructor(s): _____________________________________________________________ Please evaluate how well the following objectives were met. Rate the categories as follows: 5 - Excellent 4 - Good 3 - Satisfactory 2 - Fair 1 - Unsatisfactory Draft BD LSRII Overview Course Evaluation...
  • Page 82 Course Content Anatomy of the BD LSRII Manual Operation and Maintenance Optics and Lasers Intro to DigiFACS Software Instrument QC Sample Optimization and Acquisition Sample Analysis Data Management Overall Course Rating Please explain specifically any category rated 1 or 2.
  • Page 83 Did the course meet your expectations? Draft BD LSRII Overview Course Evaluation...
  • Page 84 BD LSRII Overview Course Evaluation Draft...

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