EIJ2_SWS30_FLO10_2_uls_beads_600s 2023-01-05 07h22.cyz ----- Processed at : 05-Jan-2023 07:39:45 ----- 1. The cytometric analysis is performed on machine LelystadII 2. The CytoUSB software used is CytoUSB Version: 5.11.1.6 3. The filename is EIJ2_SWS30_FLO10_2_uls_beads_600s 2023-01-05 07h22.cyz 4. The analysis is performed on 05-01-2023 time 07:28:12 5. The number of measured particles is 500790 6. The total concentration found (e.g. only SWS trigg.) is 3535 particles/microliter 7. The concentration found (all used triggers) is 884 particles/microliter 8. The preconcentration (before analysis) found (all used triggers) is 392 particles/microliter 9. The flush time found (between samples) is 90 seconds 10. The effective analysis volume is 566.4539 microliter 11. The pumped volume is 1218 microliter 12. The particle-rate is 7160 particles passing the laser beam/second 13. The used sample flow rate is 2.0252 microliter/second 14. The used block size memory is 64 kB 15. Trigger 1: SWS HS at threshold level 30 mV 16. Trigger 2: Maximum FL Orange HS at threshold level 10 17. Temperature sensor PMT is : 28 C 18. Temperature sensor Sheath is : 22 C 19. Temperature sensor Sheath is : 28 C 20. Pressure sensor difference is : -494 mBar 21. Pressure sensor absolute is : 430 mBar 22. Sample pump 1: Last calibration of the sample pump was : 01-Nov-2022 23. Alignment check 1 by curvature detector is 0.95794 (1=perfect) 24. Alignment check 2 by curvature detector is 98% (100%=perfect) 25. Saturated signals (flattened peaks) occur when the dynamic range of the (HS)detector is reached 26. FWS: Nr of saturated signals is 15 (0% of 500790 measured particles) 27. FWS: Maximum signal height found is 10000 mV 28. FWS: Nr of particles with signal height <10 mV is high (122613 part. (24 %)) 29. SWS HS: Nr of saturated signals is 91 (0% of 500790 measured particles) 30. SWS HS: Maximum signal height found is 5000 mV 31. SWS HS: Nr of particles with signal height <10 mV is high (37 part. (0 %)) 32. FL Yellow HS: Nr of saturated signals is 1 (0% of 500790 measured particles) 33. FL Yellow HS: Maximum signal height found is 5000 mV 34. FL Yellow HS: Nr of particles with signal height <10 mV is high (141431 part. (28 %)) 35. FL Orange HS: Nr of saturated signals is 3 (0% of 500790 measured particles) 36. FL Orange HS: Maximum signal height found is 5000 mV 37. FL Orange HS: Nr of particles with signal height <10 mV is high (0 part. (0 %)) 38. FL Red HS: Nr of saturated signals is 21 (0% of 500790 measured particles) 39. FL Red HS: Maximum signal height found is 5000 mV 40. FL Red HS: Nr of particles with signal height <10 mV is high (482268 part. (96 %)) 41. FL Yellow HS: Nr of block shaped signals is 5928 (1.1837%) 42. FL Orange HS: Nr of block shaped signals is 262 (0.052317%) 43. SWS HS: Nr of block shaped signals is 49 (0.0097845%) 44. FWS: Nr of block shaped signals is 35 (0.006989%) 45. FL Red HS: Nr of block shaped signals is 2 (0.00039937%) 46. Suspect nr: 0=low, 10=high 47. Maximum FWS: suspect = 0 48. Maximum SWS HS: suspect = 0 49. Maximum FL Yellow HS: suspect = 0 50. Maximum FL Orange HS: suspect = 0 51. Maximum FL Red HS: suspect = 0 52. Maximum SWS LS: suspect = 0 53. Maximum FL Yellow LS: suspect = 0 54. Maximum FL Red LS: suspect = 0 55. SWS HS: PMT level is 47 56. FL Yellow HS: PMT level is 88 57. FL Orange HS: PMT level is 86 58. FL Red HS: PMT level is 101 59. SWS LS: PMT level is 33 60. FL Yellow LS: PMT level is 66 61. FL Red LS: PMT level is 75 62. IIF: Imaging in Flow pictures available. Nr of images is 252 -------------------------- Warnings info -------------------------- 1.02 Warning: The number of saved particles in this analysis is quite high 1.02 Warning: Is it really necessary to store so many particles? E.g. data processing will take longer 1.02.1 Solution - Do not stop analysis after setted time, but after setted number of analyzed particles (in CytoUSB) 1.03 Warning: Your preconcentration differs more than 25% from the during analysis concentration 1.03.1 Solution - Carry over between samples? > Flush longer 1.03.2 Solution - Better homogenize samples during subsampling by a gentle stirrer 1.03.3 Solution - Colonies-filaments of cells break during analysis > stir slower 1.05 Warning: Your effective analysis volume is low compared to the pumped analysis volume 1.05.1 Solution - Set blocksize memory in CytoUSB higher, used=64k (default=4k) e.g. to 128k 1.07 Warning: Analysis with high particle rate (7160), meaning you have higher chance of coincidence 1.07.1 Solution - Decrease, use lower sample rate 1.07.2 Solution - Is threshold in noise level? Increase threshold and find out of particle rate is suddenly lower and quite stable 1.07.3 Solution - Is the sheath fluid clean enough? 1.11 Warning: This threshold level is quite high 1.11 Warning: Take care you don't miss smaller particles, because they will not be triggered at this level 1.11.1 Solution - Set lower threshold trigger level 1.11.2 Solution - If you need to put the level that high to measure something, it might be causeed by increased noise. 1.11.3 Solution - Increased noise is usually caused by dirty cuvette (inner) walls/tubing/sheath fluid - So, clean the cuvette carefully, change tubing, use new sheath 1.20 Warning: SWS LS: Maximum is 154 mV if HS is around 4500 mV and too high. 1.20.1 Solution - Put LS PMT lower. Maximum LS around 50 mV (if HS is around 4500 mV). 1.20 Warning: SWS LS: HS-LS ratio is 51 and too low. 1.20.1 Solution - Put LS PMT lower. Ratio HS/LS should be around 100. 1.20 Warning: FL Yellow LS: HS-LS ratio is 4 and too low. 1.20.1 Solution - Put LS PMT lower. Ratio HS/LS should be around 100. 1.20 Warning: FL Yellow LS: HS-LS ratio is 8 and too low. 1.20.1 Solution - Put LS PMT lower. Ratio HS/LS should be around 100. 1.20 Warning: FL Red LS: HS-LS ratio is 70 and too low. 1.20.1 Solution - Put LS PMT lower. Ratio HS/LS should be around 100. 1.23 Warning: FL Red HS : Nr of particles with signal height <10 mV is high (96 %) 1.23.1 Solution - Increase, set higher trigger threshold (to prevent measuring noise) 1.23.2 Solution - Decrease, set lower particle sample rate. Maybe caused by an overload of particles/ noise in your sample 1.23.3 Solution - Clean sheath, clean cuvette, clean tubing? (noise can be introduced by dirty walls)