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**Introduction to Flow Cytometry**

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

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The Instrument 2

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**Optics (Emission Detectors)**

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**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

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Filters 5

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Optical Layout 6

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**Detectors and Signal Processing**

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**Detection PMT Photo Multiplier Tube**

PMT’s collect photons that are then converted into voltage signals 8

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Pulse Flowing Stream Voltage pulse Laser 9

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

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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

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**Doublet Discrimination**

W 2W H W 2H H A 2A 2A FL-W FL-A Single cells doublets FL-H 12

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**Threshold Forward Scatter Threshold Voltage H Threshold Time W**

Small Cells and debris Cells of Interest 13

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Data Handling

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Histograms Cell Count Fluorescence in a cell Fluorescence

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What are those dots?

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

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**Gating Positive or Negative?**

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

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

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Dot vs Countour Plots Dot Plots Contour and Density Plots 20

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**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

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**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

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**Analysis Software FlowJo X VenturiOne CellQuest Summit FCSExpress**

Kaluza FACSDiva 23

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**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

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**Introduction to Flow Cytometry**

IGC Workshop Fundamentals of Flow Cytometry (end) Rui Gardner IGC – April 02, 2013

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