Introduction to the LSR II and FACSDiva 6.0 Software

Four-laser, 10-color benchtop flow cytometer side door left cover right cover High Throughput Sampler fluidics interconnects control panel SIP power switch The LSRII is a four laser benchtop flow cytometer.It has 12 detectors and can detect 10 colors simultaneously. It has a "Blue" (488nm) laser, a "Red" (633nm) laser, a "UV" (355nm) laser, and a "Violet" (405nm) laser. The acquisition workstation is a PC based software FACS Diva 6.0. The data files can be exported and converted for analysis using other software such as, CellQuest Pro, WinMDI and Flowjo. This unit also has a high throughput autosampler which provides automatic and rapid sample acquisition from either a 96- or 384-well plate. Laser: 488nm, 355nm, 405 nm, 633 nm

Control Panel Sample flow rates: approximately 12, 35, and 60 μL/min ( LO, MED, and HI settings ) SAMPLE FINE ADJ LO MED HI When the SAMPLE FINE ADJ knob is at its midpoint, the sample flow rates at the LO, MED, and HI settings are approximately 12, 35, and 60 μL/min of sample, respectively. RUN STNDBY PRIME sample flow rate fluid control buttons fluid control buttons

Octagon Detector Arrays longpass dichroic mirrors bandpass filters The cover of LSRII can be opened. The user can access to the dichroic filters to change the configuration of the instrument. LSRII uses fixed-alignment lasers. The scatter light or fluorescent light are transmitted by dichroic filters to octagon and trigon detectors. The octagon and trigon detector arrays use longpass filters on their inner rings, and bandpass filters on their outer rings . Each filters are user-interchangeable. This gives more flexibility of the machine and multicolor experiments are now easier when running them on the LSR II. 355 nm (UV) : Indo-1, DAPI, Alexa Fluor 350 405 nm (Violet) : CFP, Alexa Fluor 430, Pacific Blue, Marina Blue, Alexa Fluor 405 488 nm (Blue) : FITC, PE, PerCP, Pe-Cy5 (aka CyChrome), PI, PerCp-Cy5.5, PE-Cy7, GFP, PE-Texas Red 633 nm (Red): APC, APC-Cy7, Alexa Fluor 647, Alexa 680

488-nm blue laser 633-nm red laser PerCP-Cy5.5 695/40BP 685LP PerCP PI PE-Cy5 PE-Cy5 APC 670/14 660/20 FITC 635 LP 530/30 GFP 505 LP 488/10 SSC Here is the default filter settings of blue laser and red laser in our LSR. That is the most popular conbination of the dyes. 735 LP 735 LP 575/26 780/60 780/60 PE PE-Cy7 APC-Cy7

355-nm UV laser 405-nm violet laser Alexa405 DAPI CascadeB Pacific Blue Alexa350 440/40 450/50 505 LP 505 LP 525/50 530/30 AmCyan CascadeY Indo-1 (Blue)

To Login the computer Login Name: flow user Password: fccf To start the software, double click the shortcut icon on the desktop shortcut icon When loging into the computer, you can find a shortcut icon of the software on the desktop. Double click the icon. the Log-In dialog for the software appears, enter the user name and password for each user. Each research group has their own password and could be used in all our instruments. Login to FACSDiva Software

FACSDiva workspace After a successful login, the BD FACSDiva workspace appears showing the main application windows which include Browser, Cytometer, inspector, Acquistion Dashboard and global worksheet. Most software functions are controlled using the tool bar at the top of the workspace and toolbars within the Browser and Worksheet windows. Sample Acquisition is controlled by using the buttons within the Acquisition Dashboard . The Status bar at the middle or the bottom of the workspace provides cytometer connection status, fluidics information. All the windows can be hidden or shows by clicking a button on the workspace toolbar.

Preparing Sheath and Waste Containers The sheath and waste containers are outside the cytometer and are positioned on the floor. Before you start to run the sample in the machine, check the fluid levels in the sheath and waste containers to make sure that you do not run out of sheath fluid during an experiment and that the waste container does not become too full. Check the tank and the connection point, make sure there is no leakage. Prime the system and get rid of all the air bubbles. Fluidics Cleaning Each time you finish using the cytometer, clean the sample injection tube and the area between the injection tube and the outer sleeve. This prevents the sample injection tube from becoming clogged and removes dyes that can remain in the tubing. CLEANING INSTRUCTIONS ARE POSTED ON THE LSRII

BioSquare III, 670 Albany St. 5th Floor LSRII Location: BioSquare III, 670 Albany St. 5th Floor (Card access is required to enter the room) FCCF web address: http://www.bu.edu/cores/flow-cytometry FCCF email: flowcore@bu.edu Sorting facility phone number: 617-414-5225