| Content Areas |
| I. Instrumentation |
| A. Fluidics - Purdue Flow Cytometry, Lecture 9, Flow Systems and Hydrodynamics |
| 1. Hydrodynamic focusing and properties of sheath fluids |
| 2. Generation of differential pressures (e.g., syringe pump, pressure based) |
| B. Optics |
| 1. Optical filters (e.g., long pass, band pass, short pass, dichroics, neutral density, polarizing) from Flow Cytometry Theory, Optics - Filter Properties with slides modified from Doctors J.Paul Robinson and Robert Murphy |
| 2. Light source (e.g., laser type, laser line, arc lamp, led) from Purdue Flow Cytometry, Lecture 5, Light Sources & Optical Systems |
| 3. Lenses (e.g., beam shape, collecting, focusing, objective) from Flowbook, Dr. Michael Omerond, Chapter 2: The Flow Cytometer |
| 4. Optical pathway (e.g., transmission, reflection, interrogation point, collinear, spatial separation, light scatter) from Purdue Flow Cytometry, Lecture 6, Optics - Filter Properties |
| C. Electronics |
| 1. Amplifiers (e.g., Linear, Logarithmic) from Data Collection: Linear, log, ratios,” adapted from pages 163-171, Practical flow cytometry, 3rd Edition, by Howard M. Shapiro, M.D., Wiley-Liss, Inc |
| 2. Detectors (e.g., photomultiplier tube, photodiode, CCD camera, avalanche photon detector) from Purdue Flow Cytometry, Lecture 7 Flow Cytometry:Theory, Detectors & Fluidics |
| 3. Digital vs. analog systems
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| 4. Noise from Flow Cytometer Electronics Review Article, C.Snow, Cytometry Part A 57A:63–69 (2004), 2004 Wiley-Liss, Inc |
| 5. Pulse measurement (e.g., time delay, window extension, area, width, Coulter impedance) from Electronic Measurements & Signal Processing, adapted from pages 145-149, Practical flow cytometry, 3rd Edition, by Howard M. Shapiro, M.D., Wiley-Liss, Inc |
| 6. Threshold/discriminator from Introduction to Flow Cytometry: A Learning Guide, Chapter 4 “Optical Systems,” Section 4.3 “Signal Detection,” Manual Part Number: 11-11032-01 April 2000, |
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| II. Sample |
| 1. Sample source (e.g., beads, microspheres, solid tissue, body fluids, subcellular components, cultured cells, microorganisms, plants, whole organisms) from Einstein Flow Cytometry Core, Frequently Asked Questions |
| 2. Sample integrity (e.g., collection, handling, storage viability) |
| 3. Sample preparation and staining (e.g., disaggregation, lysing agents, aggregates, filtering, fixation, permeabilization) from AbDserotec.com/Support/Sample Preparation, |
| 4. Cell enrichment (e.g., cell sorting, density gradient isolation, magnetic beads) |
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| III. Data |
| 1. Data standards (e.g., image file format, FCS format, metadata, multichannel data, XML, storage requirements) |
| 2. Signal processing (e.g., binning, compensation, pulse processing, baseline restoration, background correction) |
| 3. Data display (e.g., types of displays, transformations) |
| 4. Gating (e.g., hierarchical vs. Boolean gating, gates, regions) |
| 5. Statistical methods (e.g., central tendency, standard deviation, CV, KS statistics, cluster analysis, principal component analysis, discriminant analysis) |
| 6. Common data modeling techniques (e.g., DNA ploidy, cell cycle analysis, proliferation, phenotyping, ratiometric) |
| 7. Quantitative cytometry (e.g., molecules of equivalent soluble fluorochrome [MESF], absolute counts) |
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| IV. Safety |
| 1. Biosafety procedures (e.g., biosafety categories, Personal Protective Equipment, specimen transport and preparation precautions, aerosols, decontamination)
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| 2. Instrument safety (e.g., lasers, electronics) from Flowbook, Dr. Michael Omerond, Appendix 4: Safety |
| 3. Chemical safety (e.g., mutagenic agents, cytotoxic agents) from Health and Safety Executive, Guidance, Topics, Control of Substances Hazardous to Health |
| 4. Environmental safety (e.g., waste disposal)
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| V. Quality Control |
| 1. Instrument quality control (e.g., optical alignment, detector calibration)
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| 2. Reagent quality control (e.g., panel verification, titration, lot to lot variation, storage, handling)
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| 3. Sample integrity |
| 4. Appropriate sample quality controls selection (internal, external) |
| 5. Trend analysis and interpretation |
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| VI. Experimental Design |
| A. Assay Development |
| 1. Sample state (e.g., activated, resting, proliferating) |
| 2. Target (e.g., cell type, subcellular location, molecule) |
| 3. Assay interpretation (e.g., isotype control, autofluorescence, biological systems control, background measurement controls) |
| 4. Assay optimization (e.g., appropriate use of limited sample, frequency of target, cell seeding, kinetics, scalability, blocking, signal to noise, statistical design, Z factor) |
| B. Reagent Selection |
1. Fluorochrome issues (e.g., antigen density, protein coexpression, optimal combination, photostability, F/P ratio, spectral overlap and compensation, quenching)
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| 2. Probe types (e.g., antibodies, viability/DNA dyes, physiological, tracking, fluorescent proteins) |
| 3. Solutions (e.g., buffers, fixatives, chelators, permeabilizing agents) from Institute of Cell and Molecular Science, The Blizard Building, Barts and The London School of Medicine and Dentistry |
| C. Assay Validation |
| 1. Method validation (e.g., accuracy, reproducibility/precision, sensitivity, specificity, linearity, reference range, robustness)
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| 2. Method calibration (e.g., standards, controls) from Robert A. Hoffman, Current Protocols in Cytometry (2005) 1.3.1-1.3.21, Copyright 2005 by John Wiley & Sons, Inc. |
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| VII. Theoretical Principles |
| A. Physical Principles |
| 1. Properties of light (e.g., refraction, diffraction, polarization, scatter) |
| 2. Fluorescence (e.g., Stokes shift, excitation and emission, energy transfer, environmental sensitivity [such as pH, polarity, calcium])
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| 3. Optics (e.g., optical filters, image formation, focal plane, numerical aperture) |
| 4. Electronics (e.g., signal detection, amplification, processing) |
| 5. Fluid dynamics (e.g., laminar flow, stream width, turbulence) |
| 6. Cell sorting (e.g., Jet-in-air, cuvette, droplet formation, drop delay, drop deflection, fanning, charging) |
| B. Biological Principles |
| 1. Antigen/antibody interaction and antibody structure
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| 2. Fluorescent proteins structure and properties
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| 3. Optical properties of cells |