Principles of flow cytometry: overview of flow cytometry and its uses for cell analysis and sorting Shoreline Community College BIOL 288
Flow Cytometry What is Flow Cytometry? Measurement of cells or particles in a fluid stream The first commercial cytometer was developed in 1956 (Coulter counter) The first fluorescent based cytometer was developed in 1968. In 1978 the term Flow Cytometry was adopted and companies began to manufacture commercial instrument systems
Flow Cytometry Flow cytometry uses fluorescent light and non-fluorescent light to categorize and quantify cells or particles. An Instrument collects and measures multiple characteristics of individual cells within a population as they pass through a focused beam of light. Modern flow cytometers are powerful tools; at rates of several thousand to 10 s of thousand cells per second, quantifiable data on complex mixtures can be obtained revealing the heterogeneity of a sample and its many subsets of cells. Examples of use Immunophenotyping Cell proliferation Tracking proteins or genes Activation studies Immune response Apoptosis Cell sorting All of the above and in addition, single to multiple populations are physically separated and purified from a mixed sample for downstream assays
Flow Cytometry Where is flow cytometry used? Biomedical research labs Immunology, Cancer Biology, Neurobiology, Molecular biology, Microbiology, Parasitology Diagnostic Laboratories Virology - HIV/AIDS, Hematology, Transplant and Tumor Immunology, Prenatal Diagnosis Medical Engineering Protein Engineering, Microvessicle and Nanoparticles Marine and Plant Biology
Fluorescence Fluorescent molecules emit light energy within a spectral range Fluorescent Dyes and Proteins are selective and highly sensitive detection molecules used as markers to classify cellular properties. Monoclonal antibodies and tetramers when coupled with fluorescent dyes can be detected using flow cytometry.
Process of Fluorescence Absorption Excitation Emission
Absorption Excitation The absorption wavelength is always a higher energy than the emission wavelength Emission Higher energy=shorter wavelength Lower energy=longer wavelength
Fluorochromes Excitation spectral wavelength Emission range or Laser spectral Line range
Components of a Flow Cytometer Fluidics Optics Electronics Sheath Sample Light source (lasers) Optical path (filters & mirrors) Light collection (detectors) Detector signal processing Computer interface Data storage & output
Components of a Flow Cytometer Fluorescence & Side scatter Electronics Fluidics Optics Forward scatter Focused Laser Beam Sheath fluid Sample Injector Tip
Cell Complexity Forward vs. Side Scatter Granulocyte Monocyte Debris Platelet's RBC Lymphocyte Cell Size
Optical System 488nm Blue laser Flow Cytometer Fluidic System Sheath fluid Cleaning and Waste 633nm Red laser Sample Intake Probe (SIT) FL1 FL4 Fluorescent detectors FL2 FL3 Flow Cell
Filters 530 +/- 15 {515 545} nm range 530/30 nm Filter
Common Fluorescent Markers Dye or Protein Excitation Maximum Emission Maximum Pacific Blue 405 455 Pacific Orange 405 551 BV605 405 605 PE 480;565 578 PE-Cy7 conjugates 480;565 767 PE-Texas Red 480;565 613 FITC 495 519 PerCP-Cy5.5 490 694 EGFP 484 507 EYFP 514 527 mcherry 587 610 APC 650 660 APC-Cy7 conjugates 650;755 767 Alexa Fluor 680 679 702
PE Multicolor detection Forward scatter Focused Laser Beam Sheath fluid Sample Injector Tip
Spectral Spillover PE Spillover into FITC = 0.7% FITC Spillover into PE = 22.7%
Compensation Autofluorescence PE - 15% 25% 32% FITC uncompensated
Panel Design Choose fluorochromes that are spectrally separated and spread the colors across more lasers. This decrease the amount of compensation needed. LSR II * every color in a panel needs a single color control sample for compensation
Panel Design Pair the dimmest marker to your brightest fluorochrome. Utilize the Staining Index (SI) to determine brightness of dyes. But remember minimizing spillover especially when trying to detect a dim population in combination with other highly expressed ones may require alternate dye choices
Panel Design Controls Utilize controls that are well matched to the experimental sample. Same cells type, same antibody if possible Watch out for issues with tandem dyes, i.e. PE-Cy7, APC-Cy7 Use comp beads if you don t have enough cells for your controls or if the target population is in low abundance Know the difference from instrumentation controls and experimental controls. WT vs. KO Treatment vs No treatment
Common Issues Background: Can obscure detection of target-specific antibodies. There are several types of background in flow cytometry. Autofluorescence Spectral Overlap FSC Sensor Fluorescence Minus One = FMO Non-specific binding *Breakdown of tandem dyes
Common Issues Tandem Dyes: Expand the number of colors that can be detected from a single laser source. How they work Potential issues FSC Sensor 488nm Fluorescence detector (FITC, PE etc.) PE Cy7 PE-Cy7 ex:488nm em:575nm ex:570nm em:767nm ex:488/570nm em:767nm
Cell Sorting Basic model Laser Light Source Pulse charge applied to stream when a cell to be sorted is detected FSC Charged Plates + - Sorted cell purity can be 98.5% or higher but many factors impact purity. What are they?