Overview of Flow Cytometry and Its Uses for Cell Analysis and Sorting

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 The absorption wavelength is always a higher energy than the emission Excitation wavelength

Emission

Higher energy=shorter wavelength Lower energy=longer wavelength Fluorochromes

Excitation spectralwavelengthEmission range or Laser spectral Line range Components of a Flow Cytometer

Fluidics Optics Electronics

Sheath Light source (lasers) Detector signal processing Sample Optical path (filters & mirrors) Computer interface Light collection (detectors) Data storage & output Components of a Flow Cytometer

Fluorescence ElectronicsFluidicsOptics & Side scatter

Forward scatter

Focused Laser Beam Sheath fluid Sample Injector Tip Forward vs. Side Scatter

Granulocyte

Monocyte Cell Cell Complexity

Platelet's Debris RBC Lymphocyte

Cell Size Flow Cytometer Fluidic System Sheath fluid Cleaning and Waste Optical System 488nm Blue laser

633nm Red laser Sample Intake Probe (SIT)

FL1 FL4 Fluorescent detectors

FL2 Flow Cell FL3 Filters

530 +/- 15 {515 – 545} nm range

530/30 nm Filter

Transmitted Light

660/30 nm Filter Common Fluorescent Markers

Excitation Emission Dye or Protein Maximum 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

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 PEPE -- 15%5%25%32% FITC FITCFITC

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 PE Cy7 (FITC, PE etc.)PE -Cy7 ex:488nm em:575nm ex:570nm em:767nm ex:488/570nm em:767nm Cell Sorting

• Basic model

Laser Light Source FSC Pulse charge applied to stream when a cell to be sorted is detected

Charged Plates + -

Sorted cell purity can be 98.5% or higher but many factors impact purity. What are they?