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Fundamentals of Flow Cytometry ( cont .)

Introduction to Flow Cytometry. IGC Workshop. Fundamentals of Flow Cytometry ( cont .). Rui Gardner. IGC – April 02, 2013. The Instrument. 2. Optics (Emission Detectors). Filters. Blocked. Blocked. Filtered. Filtered. Blocked. BP : Band Pass Filter. LP : Long Pass Filter.

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Fundamentals of Flow Cytometry ( cont .)

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  1. Introduction to FlowCytometry IGC Workshop Fundamentals ofFlowCytometry(cont.) Rui Gardner IGC – April 02, 2013

  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. Histograms Cell Count Fluorescence in a cell Fluorescence

  16. What are those dots?

  17. Gating Common Gate Shapes Logical Gating AND, OR, NOT 17

  18. Gating Positive or Negative? A “positive” cell or event is that which falls outside the “negative” gate. Neg Pos 18

  19. BackGating Backgating a positive populationcanenrichthepopulationofinterestandhelpidentifyitcorrectly CD4 FITC Lymphocytes CD4- CD4+ 19

  20. Dot vs Countour Plots Dot Plots Contour and Density 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. 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 22

  23. Analysis Software FlowJo X VenturiOne CellQuest Summit FCSExpress Kaluza FACSDiva 23

  24. Analysis Software Commercial Analysis Software: Open Source or Free software: FlowJo X (TreeStar) - Site License @ IGC CellQuest/Pro (BD) - 3 Licenses @ IGC FACSDiva (BD) - 2 Licenses @ IGC VenturiOne (Applied Cytometry) FCS Express (De Novo Software) GemStone (Verity Software) WinList (Verity Software) ModFit LT (Verity Software) FlowLogic (Inivai) Kaluza (Beckman Coulter) Guava Software (Merck-Millipore) Summit (Beckman Coulter) FlowCore (R/Bioconductor) Weasel Flowing Software Attune Software (Life Technologies) FCS Express (De Novo Software) Cyflogic Plate Analyzer Softwares Plate Analyzer (Free – Purdue University) Hyperview (Commercial – Intellicyt) FACSDiva (Commercial – BD) 24

  25. Introduction to FlowCytometry IGC Workshop Fundamentals ofFlowCytometry(end) Rui Gardner IGC – April 02, 2013

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