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What is Flow Cytometry ?

What is Flow Cytometry ?. Flow Cytometry. Introduction to Flow Cytometry. IGC Workshop. uic. Rui Gardner (IGC) Telma Lopes (IGC ) Carlos Tadokoro (IGC ). Fundamentals of Flow Cytometry ( cont .). Rui Gardner. IGC – December 15 , 2009. The Instrument. 2.

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What is Flow Cytometry ?

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  1. WhatisFlowCytometry? Flow Cytometry Introduction to Flow Cytometry IGC Workshop uic Rui Gardner (IGC) Telma Lopes (IGC) Carlos Tadokoro (IGC) Fundamentals ofFlowCytometry(cont.) Rui Gardner IGC – December 15, 2009

  2. The Instrument 2

  3. Optics(Emission Detectors)

  4. Filters Blocked Blocked Filtered Filtered Blocked BP : Band Pass Filter LP : Long Pass Filter 530 / 60 > 500 4

  5. Filters 5

  6. Optical Layout 6

  7. Detectors and Signal Processing

  8. Detection PMT Photo Multiplier Tube PMT’s collect photons that are then converted into voltage signals 8

  9. Pulse Flowing Stream Voltage pulse Laser 9

  10. Pulse Parameters W H A H: A: W: 10

  11. Height vs Area H H A A For non spherical cells, Height (FL-H) is not an adequate parameter to analyze Area (FL-A) is the most adequate. However, we still need to remove doublets from the analysis... 11

  12. Doublet Discrimination W W 2W 2H H A 2A 2A H FL-W FL-H doublets Single cells doublets Single cells FL-A FL-A 12

  13. Threshold Forward Scatter Threshold Voltage H W Threshold Time Small Cells and debris Cells of Interest 13

  14. Data Handling

  15. Analysis Software Flowjo VenturiOne CellQuest Summit FCSExpress Kaluza FACSDiva 15

  16. Gating Common Gate Shapes Logical Gating AND, OR, NOT 16

  17. Gating Positive or Negative? A “positive” cell or event is that which falls outside the “negative” gate. Neg Pos 17

  18. BackGating Backgating a positive populationcanenrichthepopulationofinterestandhelpidentifyitcorrectly CD4 FITC 18

  19. Acquisition How many cells should I acquire? Precision Counting cells follows Poisson statistics: 10,000 1002 sd 1 cv % = = cv % = N = = 400 x 100 x 100 (cv %)2 25 mean N is the number of cells counted 40,000 Example: Population of interest is 1% of total population and want 5% precision Number of cells to be counted in the region of interest Number of total cells to be counted 19

  20. Dot vs Countour Plots Contour Plots Dot Plots 20

  21. Logarithmic or Linear? Anti-CD4-labeled antibody Signals vary >100-fold Use Log scale Linear Log DNA-labeling dye Signals vary 2- to 10-fold Use Linear scale Linear Log 21

  22. Logicle (Bioexponential) Transformation Taken from Herzenberg, et. al (2006) “Interpreting flow cytometry data: a guide for theperplexed”, Nat Immunol, 7:681-685 22

  23. LogicleTransformation(compensation) Taken from Herzenberg, et. al (2006) “Interpreting flow cytometry data: a guide for theperplexed”, Nat Immunol, 7:681-685 23

  24. WhatisFlowCytometry? Flow Cytometry Introduction to Flow Cytometry IGC Workshop uic Rui Gardner (IGC) Telma Lopes (IGC) Carlos Tadokoro (IGC) Fundamentals ofFlowCytometry(end) Rui Gardner IGC – November 9, 2009

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