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Fundamentals of Flow Cytometry (cont.) IGC – April 02, 2013 Introduction to Flow Cytometry IGC Workshop.

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Presentation on theme: "Fundamentals of Flow Cytometry (cont.) IGC – April 02, 2013 Introduction to Flow Cytometry IGC Workshop."— Presentation transcript:

1 Fundamentals of Flow Cytometry (cont.) IGC – April 02, 2013 Introduction to Flow Cytometry IGC Workshop

2 The Instrument 2

3

4 FilteredBlocked BP : Band Pass Filter 530 / 60 FilteredBlocked LP : Long Pass Filter > 500 Filters 4

5 5

6 Optical Layout 6

7

8 Detection 8 PMT Photo Multiplier Tube PMTs collect photons that are then converted into voltage signals

9 Pulse 9 Laser Voltage pulse Flowing Stream

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

11 Height vs Area 11 H H AA 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...

12 Doublet Discrimination 12 2W H W 2H W H A2A FL-W FL-A Single cells doublets FL-H FL-A Single cells doublets

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

14

15 Histograms Fluorescence Cell Count Fluorescence in a cell

16 What are those dots?

17 Gating 17 Common Gate ShapesLogical Gating AND, OR, NOT

18 Gating 18 A positive cell or event is that which falls outside the negative gate. Positive or Negative? PosNeg

19 Back Gating 19 Back gating a positive population can enrich the population of interest and help identify it correctly CD4 FITC Lymphocytes CD4- CD4+

20 Dot vs Countour Plots 20 Dot PlotsContour and Density Plots

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

22 Acquisition 22 How many cells should I acquire? Counting cells follows Poisson statistics: cv % = sd mean x 100 N is the number of cells counted Precision cv % = 1 x 100 Example: Population of interest is 1% of total population and want 5% precision N = (cv %) 2 = 10, = ,000 Number of cells to be counted in the region of interest Number of total cells to be counted Number of total cells to be counted

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

24 Analysis Software 24 Commercial Analysis Software: FlowJo X (TreeStar) - Site IGC CellQuest/Pro (BD) - 3 IGC FACSDiva (BD) - 2 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) Open Source or Free software: 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)

25 Fundamentals of Flow Cytometry (end) IGC – April 02, 2013 Introduction to Flow Cytometry IGC Workshop


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