The Danish Stem Cell Center (DanStem) Danish Stem Cell Center Flow Cytometry Core Facility. LSR Fortessa Guide

Transcription

1 Danish Stem Cell Center Flow Cytometry Core Facility LSR Fortessa Guide

2 LSR Fortessa Overview The Danish Stem Cell Center (DanStem)

3 BD FACSFlow Supply System Fluidics Cart Sheath container Waste container

4 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

5 Opera5ng Instruc5ons The Danish Stem Cell Center (DanStem)

6 1. Turn on or restart the computer. The Danish Stem Cell Center (DanStem)

7 2. Turn on the LSRFortessa by pressing the green bujon on the side.

8 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.

9 The Danish Stem Cell Center (DanStem) 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 Saline Filter

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

11 6. Run filtered dh2o on HIGH for 5 minutes. The Danish Stem Cell Center (DanStem)

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

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

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

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

16 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.

17 13. When prompted for CST mismatch, click Use CST Sehngs.

18 14. In the top le` corner of the screen, select View and make sure that Browser, Cytometer, Inspector, Worksheet, Dashboard and Editor are checked.

19 15. Arrange the windows as preferred. Worksheet Browser Dashboard Cytometer Inspector Editor

20 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.

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

22 18. Adjust page size by clicking Page Setup under File in the taskbar. Use these sehngs to maximize page usage for Click OK.

23 19. Rename the experiment with Your plus the date (YI_DDMMYYYY) in the Browser menu by right- clicking on your experiment and Rename.

24 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.

25 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.

26 23. Rename the Specimen and Tube accordingly by right- clicking and Rename.

27 24. the first tube by clicking on the arrow on the le` side of the Tube icon. Upon the arrow will turn green. 25. You can only a tube when the experiment is loaded. A`er the Dashboard will become ac@ve.

28 26. Create your plots using the icons in the Global Worksheet toolbar.

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

30 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.

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

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

33 31. Create your gates as desired using the tools in the Global Worksheet toolbar.

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

35 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 Dashboard, followed by Record Data. Recording will stop when your stopping gate are met. You can stop acquiring or recording by clicking Stop Acquiring.

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

37 35. Acquire, record, and repeat with your next samples. The Danish Stem Cell Center (DanStem)

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

39 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.

40 38. 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.

41 39. In the Export Experiments dialog, uncheck Delete experiments a`er export when The Danish Stem Cell Center (DanStem)

42 40. You can import your experiments by to File > Import > Experiments. Find the folder of your exported experiment and select Import.

43 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 dh 2 O for 5 minutes If someone is scheduled immediately a`er you: - run filtered dh 2 O for 5 minutes Filtered dh 2 O FACSRinse

44 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!

45 If you are unsure of what to do AND if you have any PLEASE do not hesitate to ask : Gelo: gelo.delacruz@sund.ku.dk Paul: paul.dieken@cpr.ku.dk

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

47 Appendix A LSR Fortessa Configura@on Violet laser (405 nm)

48 Appendix A LSR Fortessa Configura@on Yellow- Green laser (561nm)

49 Appendix A LSR Fortessa Configura@on UV laser (355 nm) Red laser (640 nm)

50 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, 510/20 488/10 7-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

51 Appendix B 405nm A B C D E F 630LP, 670/30 600LP, 610/20 570LP, 586/15 535LP, 540/30 505LP, 525/50 450/50 Qdot655 Qdot605 Qdot585 Pacific Orange AlexaFluor430 AlexaFluor405 Qdot565 AmCyan Cascade Blue ECFP Cerulean mcfp DyLight405 T-Sapphire Pacific Blue TagCFP SYTOX Blue

52 Appendix B 640nm A B C 750LP, 780/60 690LP, 730/45 670/14 AlexaFluor750 AlexaFluor700 AlexaFluor633 AlexaFluor790 DyLight680 AlexaFluor647 APC-Cy7 APC APC-eFluor780 DRAQ5 APC-H7 DyLight633 DyLight649 TO-PRO-3 TOTO-3

53 Appendix B 561nm A B C D E 750LP, 780/60 685LP, 710/50 635LP, 670/30 600LP, 610/20 586/15 PE-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

54 Appendix B 355nm (standard) 355nm (Side Population) A B A B 505LP, 530/30 450/50 620LP 440LP, 450/65 Indo-1 (Blue) AlexaFluor350 Hoechst Red Hoechst Blue DAPI DyLight350 EBFP EBFP2 Hoechst33258 Hoechst33342 Indo-1 (Violet)