lsr fortessa guide

The Danish Stem Cell Center (DanStem) The Danish Stem Cell Center (DanStem)

LSR Fortessa Guide

Danish Stem Cell Center Flow Cytometry Core Facility

The Danish Stem Cell Center (DanStem)

LSR Fortessa Overview


BD FACSFlow™ Supply System Fluidics Cart

Sheath container Waste container


LSRFortessa Overview

•  5 dis@nct laser lines (488 nm, 405 nm, 355nm, 640 nm, 561 nm)

•  18 parameters plus scaJer •  HTS module for plate analysis (opera@ng procedure in separate

guide)

See Appendix A for the op@cal configura@on


Opera5ng Instruc5ons


1.  Turn on or restart the computer.


2.  Turn on the LSRFortessa by pressing the green buJon on the side.


3.  Check the sheath and waste tanks. If the sheath is empty, replace with a new FACSFlow box. Use the empty container as waste. Label accordingly. On the newly removed full waste container, please place a post-‐it saying “Haztabs please”.


4.  Check saline filter for air bubbles and vent if necessary. Vent by scrolling down the control wheel. Make sure the sheath fluid is flowing when ven@ng.

Saline Filter


5.  Prime the machine 3x without any tube installed by pressing “PRIME”. The machine will go on standby mode a`er each prime.


6.  Run filtered dH2O on HIGH for 5 minutes.


7.  AGAIN, check the sheath and waste levels. We do not want the machine to take up air.


8.  Log-‐in to Windows using the Administrator account and BDIS#1 as password.


9.  Start the BD FACSDiva so`ware program by double-‐clicking on the icon on the desktop.


10. Log in to the FACSDiva so`ware by selec@ng your username in the dropdown menu under “User Name”, filling in your Password and clicking “OK”.


11. Wait up to 5 minutes for the so`ware to connect to the LSR Fortessa.

12. Wait for the status bar on the boJom right of the window reads “Connected”:

–  So`ware is connec@ng: –  So`ware has successfully connected:

•  TROUBLESHOOTING TIP: If the software cannot connect after 5 minutes: –  Shut down both the computer and LSRFortessa, wait 1 minute, start the

computer, wait 1 minute, then start up the LSR Fortessa and try to connect again in FACSDiva. If the problem persists: contact Gelo or Paul.


13.  When prompted for CST mismatch, click “Use CST Sehngs”.


14.  In the top le` corner of the screen, select “View” and make sure that “Browser”, “Cytometer”, “Inspector”, “Worksheet”, “Acquisi@on Dashboard” and “Biexponen@al Editor” are checked.


15.  Arrange the windows as preferred.

Worksheet Browser Acquisi@on Dashboard

Cytometer Inspector Biexponen@al Editor


16.  Create a new experiment by clicking “New Experiment” under “Experiment” on the taskbar. Alterna@vely, you can click on the “New Experiment” icon on the Browser.


17. When prompted, select “Blank Experiment” under Experiment Templates and click “OK”. If no prompt comes up, proceed to the next step.


18.  Adjust page size by clicking “Page Setup” under “File” in the taskbar. Use these sehngs to maximize page usage for prin@ng. Click “OK”.


19. Rename the experiment with Your Ini@als plus the date (YI_DDMMYYYY) in the Browser menu by right-‐clicking on your experiment and selec@ng “Rename”.


20.  Click on Cytometer Sehngs in the Browser. In the Inspector window, delete all parameters that you do not need.

See Appendix B for a list of fluorochromes and detectors. This list can also be found next to the cytometer.


21. With your Experiment loaded, add a specimen by clicking on the “Add Specimen” icon on the Browser toolbar.

22. View the tubes under the specimen by clicking on the + next to the specimen name.


23. Rename the Specimen and Tube accordingly by right-‐clicking and selec@ng “Rename”.


24. Ac@vate the first tube by clicking on the arrow on the le` side of the Tube icon. Upon ac@va@on, the arrow will turn green.

25. You can only ac@vate a tube when the experiment is loaded.

A`er ac@va@on, the Acquisi@on Dashboard will become ac@ve.


26. Create your acquisi@on plots using the icons in the Global Worksheet toolbar.


27. Before running your samples, filter all your samples through a Celltrics 30 (#04-‐004-‐2316) filter or using the Falcon tubes with cell strainer cap (#352235).

Celltrics 30 filter

Falcon tubes with cell strainer cap


28.  Install your sample tube. Make sure you are using the right tubes (#352052) and that they are not broken or cracked. Also, make sure the arm is seated back under the tube properly.


29. Press “RUN” on the machine, and select your acquisi@on speed (LO, MED, HI). For cell cycle analysis, always run on LO.


30.  Click “Acquire Data” on the Acquisi@on Dashboard to acquire some data for ga@ng. Click “Stop Acquiring” a`erwards. Make sure you remove your tube when you are not acquiring data.


31. Create your gates as desired using the Ga@ng tools in the Global Worksheet toolbar.


32. On the Acquisi@on Dashboard, set your stopping gate and number of events to record.


33.  To acquire and record data, make sure your Sample Tube is placed correctly, the LSRFortessa is set on “RUN”, click on “Acquire Data” in the Acquisi@on Dashboard, followed by “Record Data”. Recording will stop when your stopping gate condi@ons are met. You can stop acquiring or recording by clicking “Stop Acquiring”.


34. A`er acquiring and recording your sample, click “Next Tube” to move on to the next sample.


35.  Acquire, record, and repeat with your next samples.


36.  A`er acquiring your data: Install a tube of FACSClean and run for 5 minutes on HIGH.

WARNING! Contains bleach.


37.  Export your data as FCS files to a USB s@ck/external hard drive. DO NOT DELETE YOUR FILES!!! We will delete it a`er two months.


38. Alterna@vely, you can export as Experiment and store your experiment indefinitely on your own hard disk.

Also, if you need to reuse the experiment, you can export it as an Experiment Template.


39.  In the “Export Experiments” dialog, uncheck “Delete experiments a`er export” when expor@ng.


40.  You can import your experiments by naviga@ng to File > Import > Experiments. Find the folder of your exported experiment and select “Import”.


41. A`er running FACSClean for 5 minutes: If you are the last user of the day: -‐run FACSRinse for 5 minutes, followed by filtered dH2O for 5 minutes If someone is scheduled immediately a`er you: -‐run filtered dH2O for 5 minutes

Filtered dH2O FACSRinse


42. A`er running dH2O, leave the tube installed and place the machine on “STANDBY”.

43. Log out or exit FACSDiva.

44.  If you are the last user of the day, turn off the machine by pressing the ON/OFF buJon.

NEVER leave the machine una-ended on “RUN”. This can damage the machine! Make sure the machine is on “STANDBY” or turned OFF before leaving the room!


If you are unsure of what to do AND if you have any ques@ons:

PLEASE do not hesitate to ask :

Gelo: [email protected] Paul: [email protected]


Appendix A – LSR Fortessa Configura@on Blue laser (488 nm)


Violet laser (405 nm)

Appendix A – LSR Fortessa Configura@on


Yellow-‐Green laser (561nm)



UV laser (355 nm) Red laser (640 nm)



Appendix B

448 nm (standard) 488nm (GFP/YFP discrimination)A B C A B C

685LP, 710/50 505LP, 530/30 488/10 525LP, 542/27 505LP, 510/20 488/107-AAD (488) AlexaFluor488 SSC YFP GFP SSC

PerCP AlexaFluor500 PerCP-Cy5.5 Calcein

PI (488) CFSE TruRed Cy2

Dronpa-Green DyLight488 Emerald FITC GFP, EGFP mCitrine SYTOX Green SytoxGreen TO-PRO-1 Topaz TOTO-1 TurboGFP Venus YFP, EYFP YOYO-1


Appendix B

405nmA B C D E F

630LP, 670/30 600LP, 610/20 570LP, 586/15 535LP, 540/30 505LP, 525/50 450/50Qdot655 Qdot605 Qdot585 Pacific Orange AlexaFluor430 AlexaFluor405

Qdot565 AmCyan Cascade Blue

ECFP Cerulean

mCFP DyLight405

T-Sapphire Pacific Blue

TagCFP SYTOX Blue


Appendix B

640nmA B C

750LP, 780/60 690LP, 730/45 670/14AlexaFluor750 AlexaFluor700 AlexaFluor633

AlexaFluor790 DyLight680 AlexaFluor647

APC-Cy7 APC

APC-eFluor780 DRAQ5

APC-H7 DyLight633

DyLight649

TO-PRO-3

TOTO-3


Appendix B

561nmA B C D E

750LP, 780/60 685LP, 710/50 635LP, 670/30 600LP, 610/20 586/15PE-Cy7 PE-Cy5.5 7-AAD (561) AlexaFluor568 AlexaFluor546

Cychrome AlexaFluor594 AlexaFluor555

mKate2 DyLight594 DsRed

mPlum J-Red DyLight549

nKate mCherry mOrange

PE-Cy5 mRFP1 PE

mStrawberry Rhodamine

PE-TexasRed tdTomato

TurboRFP


Appendix B

355nm (standard) 355nm (Side Population)A B A B

505LP, 530/30 450/50 620LP 440LP, 450/65Indo-1 (Blue) AlexaFluor350 Hoechst Red Hoechst Blue

DAPI

DyLight350

EBFP

EBFP2

Hoechst33258

Hoechst33342

Indo-1 (Violet)

lsr fortessa guide

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