How to Get the Best Performance out of your CyTOF

1. How to Get the Best Performance out of your CyTOF Carina Torres CyTOF User Group Meeting CYTO 2013 23 May 2013

2. Outline • History • CyTOF Workflow • Instrument Startup • Optimization • Running Samples • Instrument Shutdown • Troubleshooting • Progress Report…1 ½ years later

3. CyTOF at Lilly A group of Lilly Senior Research Scientists went to a Garry Nolan talk CyTOF installed, placed in Flow Core Experiments began 2010 2011 2012 2013 September Purchased CyTOF (#9) Panel Validation Phenotyping Data analysis Bendall, S.C., et al. Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum. Science 332 (6030): 687-696, 2011.

4. Preparing your Lab

5. Daily Workflow: Instrument Startup Check lights on instrument Turn Heater on Instrument setup Card Cage Tab Click Heater on 200oC Make sure Argon is on ▪ 100 psi at wall ▪ 50 psi on instrument Start Syringe pump (HPLC) Prepare Nebulizer and Syringe Pump Start Plasma

6. Instrument Startup CLOSE VG2 VG1 TP2 TP1 Argon BP Ex Vent

7. Instrument Startup Check lights on instrument Turn Heater on Instrument setup Card Cage Tab Click Heater on 200oC Make sure Argon is on ▪ 100 psi at wall ▪ 50 psi on instrument Start Syringe pump (HPLC) Prepare Nebulizer and Syringe Pump Start Plasma

8. Instrument Startup 20 min

9. Instrument Startup Check lights on instrument Turn Heater on Instrument setup Card Cage Tab Click Heater on 200oC Make sure Argon is on ▪ 100 psi at wall ▪ 50 psi on instrument Start Syringe pump (HPLC) Prepare Nebulizer and Syringe Pump Start Plasma

10. Instrument Startup DI H20

11. Instrument Startup Check lights on instrument Turn Heater on Instrument setup Card Cage Tab Click Heater on 200oC Make sure Argon is on ▪ 100 psi at wall ▪ 50 psi on instrument Start Syringe pump (HPLC) Prepare Nebulizer and Syringe Pump Start Plasma

12. Instrument Startup • Chiller turns on • Start-up sequence • Plasma ignites • Chiller turns on • Start-up sequence • Plasma ignites

13. Instrument Startup

14. Instrument Startup Check lights on instrument Turn Heater on Instrument setup Card Cage Tab Click Heater on 200oC Make sure Argon is on ▪ 100 psi at wall ▪ 50 psi on instrument Start Syringe pump (HPLC) Prepare Nebulizer and Syringe Pump Start Plasma

15. Instrument Startup

16. Optimization: Performance Check and Tuning ►To get the best resolution between detection channels ►Makes sure TOF values for CS and IR are <5% of peak height, calibration doesn’t typically need to be changed Mass Calibration ►Optimize to ensure efficient detection of ions ►The dual count coefficient is calculated to generate the dual signal scale that is used to quantify ion signals ►Measure sensitivity by doing a dual counts tests to ensure 159Tb dual counts are >100,000 ►Check Oxide ratio as an indication of plasma robustness. The ratio of 155Gd dual count /159Tb dual count should be less than 3%

17. Tuning Solution Optimization: Performance Check and Tuning

18. Tuning Solution MassResolution Optimization: Performance Check and Tuning Tb >400 Example: 10425/6(2)= 868 Peak height: x= 10425 (TOF) y= 525,000 (intensity) ½ max signal intensity =6

19. Tuning Solution MassResolution Mass Calibration Optimization: Performance Check and Tuning Ir Cs 9537 11457 <5% of peak height <5% of peak height 11457 9537

20. Tuning Solution MassResolution Mass Calibration Detector gain Optimization: Performance Check and Tuning All Tb 629242 136495 Max signal intensity Tb pulse counts = 4.61 (4-5)

21. Tuning Solution MassResolution Mass Calibration Detector gain Optimization: Performance Check and Tuning Dual Count Calibration Dual Count test Oxidation All 155Gd dual count 159Tb dual count =<3% 159Tb dual count >100,000 2754 218626 =1.26% CYTOF is Ready!

23. Automated QC at Lilly QC Table Pulse Dual Mass Mass PPM Counts Counts Intensity Error Error

24. Automated QC at Lilly QC Table Pulse Dual Mass Mass PPM Counts Counts Intensity Error Error QC Table Pulse Dual Mass Mass PPM Counts Counts Intensity Error Error

25. Automated QC at Lilly Dual count calibration Dual counts plot

26. When Specs Are Not Met Observation Possible Solution Parameter <400 Call for service ToF values for Cs and Ir at beginning of peak are >5% of peak height Verify ToF values and enter new values in the mass calibration tab Mass Calibration 159Tb ratio is<4 or >5 optimize detector voltage Check X-Y alignment Optimize gas flows 159Tb dual count <100,000 Optimize current Swap nebulizer 155Gd dual count 159Tb dual count >3% Optimize gas flows

27. When Specs Are Not Met Observation Possible Solution Parameter <400 Call for service ToF values for Cs and Ir at beginning of peak are >5% of peak height Verify ToF values and enter new values in the mass calibration tab Mass Calibration 159Tb ratio is<4 or >5 optimize detector voltage Check X-Y alignment Optimize gas flows 159Tb dual count <100,000 Optimize current Swap nebulizer 155Gd dual count 159Tb dual count >3% Optimize gas flows

28. When Specs Are Not Met Observation Possible Solution Parameter <400 Call for service ToF values for Cs and Ir at beginning of peak are >5% of peak height Verify ToF values and enter new values in the mass calibration tab Mass Calibration 159Tb ratio is<4 or >5 optimize detector voltage Check X-Y alignment Optimize gas flows 159Tb dual count <100,000 Optimize current Swap nebulizer 155Gd dual count 159Tb dual count >3% Optimize gas flows

29. Verify Mass Calibration Cs Ir 9537 11457 <5% of peak height <5% of peak height 9533 11453

30. When Specs Are Not Met Observation Possible Solution Parameter <400 Call for service ToF values for Cs and Ir at beginning of peak are >5% of peak height Verify ToF values and enter new values in the mass calibration tab Mass Calibration 159Tb ratio is<4 or >5 optimize detector voltage Check X-Y alignment Optimize gas flows 159Tb dual count <100,000 Optimize current Swap nebulizer 155Gd dual count 159Tb dual count >3% Optimize gas flows

31. Optimize Detector Voltage . . . . . . . . . . . . . . .

32. When Specs Are Not Met Observation Possible Solution Parameter <400 Call for service ToF values for Cs and Ir at beginning of peak are >5% of peak height Verify ToF values and enter new values in the mass calibration tab Mass Calibration 159Tb ratio is<4 or >5 optimize detector voltage Check X-Y alignment Optimize gas flows 159Tb dual count <100,000 Optimize current Swap nebulizer 155Gd dual count 159Tb dual count >3% Optimize gas flows

33. When Specs Are Not Met Observation Possible Solution Parameter <400 Call for service ToF values for Cs and Ir at beginning of peak are >3% of peak height Verify ToF values and enter new values in the mass calibration tab Mass Calibration 159Tb ratio is<4 or >5 optimize detector voltage Check X-Y alignment Optimize gas flows 159Tb dual count <100,000 Optimize current Swap nebulizer 155Gd dual count 159Tb dual count >3% Optimize gas flows

34. X-Y Alignment of the Interface All

35. Optimize Gas Flows All

36. Optimize Current All

37. Re-Check QC Mass Calibration Dual Counts Plot

38. Running Samples: User Guidelines • Make sure sample loop is clean: run H20, check Mass Cal screen to verify

39. Running Samples: User Guidelines 600 30 10

40. Running Samples: User Guidelines • Filter samples and count cells. Resuspend cells to achieve collection rate • of ideally 300-500 events/sec • Clean with H20 between samples to minimize carryover • Clean instrument when done running samples (Wash solution and H20)

41. Instrument Shutdown

42. Instrument Shutdown

43. Instrument Shutdown • Turn off HPLC • Remove nebulizer and store in 3% Citranox

44. Troubleshooting: Common Issues Cause Solution Problem 1. Plasma won’t ignite Turn it on Argon off Use Ultra High Purity Argon Argon not pure enough Double check gas Incorrect Gas? Localize source Air Leak Plasma ignites Look for air leak in the area of sample introduction Plasma does not ignite Check the torch, injector, coil for condensation

45. Troubleshooting: Common Issues Cause Solution Problem 2. No/low signal Sample line capillary pinched Inspect capillary line Sample line capillary inserted improperly Reinsert capillary Check spray/swap nebulizer Clogged nebulizer Check gas flows Insufficient gas flows

46. Troubleshooting: Unusual Circumstances Cause Solution Problem 1. • Pankaj came out • and found the • leak. Replaced • torch and replaced • RFG coil • Leak in one • of the argon • lines • Arcing because • the coil not • aligned • Oxidation on • the coils • Clean/Replace • coil

47. Troubleshooting: Unusual Circumstances Cause Solution Problem 2. Poor contact of skimmer/reducer with the contact ring on the interface. This may be due to oxidation on the contact ring or on the reducer itself. Vigorously cleaned the contact ring on the interface with methanol. Eventually, Daniel came out to replace the interface

48. Troubleshooting: Unusual Circumstances Cause Solution Problem 3. Originally thought it was a faulty syringe pump. Turned out to be a bad valve. Replaced the entire valve assembly

49. Troubleshooting: Unusual Circumstances Cause Solution Problem 4. Clean/swap nebulizer Partially clogged/dirty nebulizer Sample capillary not positioned properly Reinsert capillary Bad syringe Replace syringe

50. Troubleshooting: Unusual Circumstances Cause Solution Problem 5. Cleaned ignitor pin with sandpaper Tungsten contamination; but from where? 180-190 180-190