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Introduction to Flow Cytometry

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Presentation on theme: "Introduction to Flow Cytometry"— Presentation transcript:

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

2 The Instrument 2

3 Optics (Emission Detectors)

4 Filters LP : Long Pass Filter > 500 BP : Band Pass Filter 530 / 60
Filtered Blocked LP : Long Pass Filter > 500 Filtered Blocked BP : Band Pass Filter 530 / 60 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 2W H W 2H H A 2A 2A FL-W FL-A Single cells doublets FL-H 12

13 Threshold Forward Scatter Threshold Voltage H Threshold Time W
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 Back Gating Back gating a positive population can enrich the population of interest and help identify it correctly 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 cv % = sd mean
x 100 Counting cells follows Poisson statistics: cv % = 1 x 100 N is the number of cells counted Example: Population of interest is 1% of total population and want 5% precision N = 1002 (cv %)2 = 10,000 25 = 400 40,000 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 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) 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 Flow Cytometry
IGC Workshop Fundamentals of Flow Cytometry (end) Rui Gardner IGC – April 02, 2013

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