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Cell Methods
Molecular Diagnostics: BIO 2064

Immunohistochemistry
• Tissue can be fixed in Paraformaldehyde to preserve the cells and biomolecules,
including proteins
• Protein biomarker is detected through primary antibody specific for the biomarker
• Signal is detected through secondary antibody –conjugated to enzyme, and the
secondary antibody binds to the primary antibody
• Signal is detected when the enzyme converts the substrate to a color complex
• After preparation, samples can be stored for long period of time. However,
detected signal suffers from poor resolution
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Cell Methods
Molecular Diagnostics: BIO 2064

Immuno-Fluorescence

• Primary and secondary antibodies are used to identify biomarker produced by the human cells
• Secondary antibody is conjugated to fluorophore which emits fluorescence signal
• The sample cannot be stored for long period of time due to deterioration of fluorescence signa
• However, fluorescence signal provides better resolution when compared to
immunohistochemistry

Immunofluorescence staining of the von-

Willebrand-Factor (vWF) in endothelial cells
(HUVECs). Actin was stained using phalloidin
(green), nuclei are stained with DAPI (blue).
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Molecular Diagnostics: BIO 2064

Introduction To Flow Cytometry
• What is Flow Cytometry?
• Components of Flow Cytometry
• How the components work?
• Collection of data through Flow Cytometry
• Applications of Flow Cytometry
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What is Flow
Molecular Cytometry
Diagnostics: BIO 2064 ?

•Flow: Fluid
•Cyto: “Cell”
•Metry: “Count/Measurement”
• It is an instrument used to measure cells in liquid suspension
• In addition, it can be used to measure other particles,
including chromosomes, bacteria, and beads
• Particle sizes ranging from 0.2 um to 15 um can be analyzed
• 1µm (micrometer)=1x10-6 meter
• Particle sizes (examples):
a) Bacteria (0.5µm)
b) Red Blood Cell (6µm)
c) Lymphocyte (8µm)
d) Neutrophil (12µm)
e) Monocyte (14µm)
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Molecular Diagnostics: BIO 2064

Components of Flow Cytometer
• Fluidics (liquid handling)
• Optics (laser beams, and photo-receptor)
• Electronics (conversion of photon to electrical signal)
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Molecular Diagnostics: BIO 2064

Fluidic System of Flow Cytometer
• Tissues or biopsy samples must be dissociated into single cell suspension
• Cells must be re-suspended in suitable solution such as buffer (PBS, pH7.5)
• For cell isolation or sorting, must practice sterile techniques, and use filtered
or autoclaved solution

Cell Suspension

Sample port
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Flow Cytometer Optic System

Molecular Diagnostics: BIO 2064
• Laser beams (blue , red, or violet)
are focused by lenses and directed toward sample flow cells
• The lights that are deflated or scattered by the samples are
collected as FSC and SSC
• For samples that are fluorescently labeled with fluorophore, laser beams “excite” the fluorophore,
leading to emission of fluorescent light which passes through collection lens, filter,
and detected by light sensitive detector (photo-detection)
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Molecular
Hydrodynamic Diagnostics:
Focusing and FlowBIO 2064
Rate

Hydrodynamic Focusing Effect of Flow Rate (events/second)

• Low flow rate (low sample pressure) through sample core: One cell at a time when passing through laser beam
• High flow rate (high sample pressure) through sample core: more than one cell may pass through the laser beam
• High flow rate may decrease data resolution

http://medicine.umich.edu/medschool/sites/medicine.umich.edu.medschool/files/
research_brcf_flowcytometry_training_cytometersubsystems_fluidics_typicalflowcells1.gif
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FSC and SSC

Molecular Diagnostics: BIO 2064

• Forward scattered light (FSC): Determines by particle size, and refractive index

FSC

Particle size FSC

Difference
Refractive Index Comparison: in IR FSC
(compare to sheath fluid)
1. Water: 1.33
2. Quartz: 1.54
3. Diamond: 2.41
http://www.usfacetersguild.org/articles/bob_keller/refractive_index/
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Molecular Diagnostics: BIO 2064

SSC: Depends on Granularity or complexity of particles

Granularity SSC
SSC

Laser

SSC
Laser

SSC
Laser
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Molecular Diagnostics: BIO 2064

FSC and SSC by cell type
Size: 6µm Size: 8µm Size: 12µm

Red Blood Cell Lymphocyte Neutrophil

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Molecular
PlottingDiagnostics:
FSC and BIOSSC2064
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Molecular Diagnostics: BIO 2064

Flow Cytometry: Fluorescence Detection of Cell surface markers

•Antibody is conjugated to Fluorophore (i.e FITC)

•Antibody binds to cell surface protein marker (i.e CD3 of T Cell)

FITC signal
Auto-Fluorescence
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Molecular Diagnostics: BIO 2064

Multi-color Analysis of cell surface markers: CD3+, and CD4+ (T Cells)

• Anti-CD3 antibody is conjugated to APC fluorophore

• Anti-CD4 antibody is conjugated to PE fluorophore
• Both antibodies are added to cell population
• Binding of conjugated antibodies to cell surface
protein markers would label cells with fluorescence
• Fluorescence labels are sorted, and displayed as digital signal
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Molecular Diagnostics: BIO 2064

Clinical and Scientific applications of flow Cytometry
• Clinical applications:
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Flow Cytometry analysis

Molecular of AcuteBIO
Diagnostics: Myeloid
2064 Leukemia

Acute Myeloid Leukemia (CD13+ and CD 34+ Myeloid cells)

http://www.clinchem.org/content/46/8/1221.full
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Molecular Diagnostics: BIO 2064

JC-1ataining of cells undergoing apoptosis

•Apoptotic cells progressively losing mitochondrial membrane potentials

•Apoptotic cells stained with JC-1 dye that emits green fluorescence

http://www.lifetechnologies.com/potential-probe.html
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Summary
Molecular Diagnostics: BIO 2064

• Flow cytometry is used to measure cells and particles based on FSC and SSC
• FSC measures particle size and refractive index of particles
• SSC measures granularity or complexity of cell content
• Fluorescently labeled cells and DNA can be quantitatively analyzed
• Clinical applications of flow cytometry includes identification of cancers,
and HIV infections
 Review Questions
1. What properties of a cell or particle can be measured by a flow cytometer?

2. What light source is used in most flow cytometers?

3. What are the three main systems in a flow cytometer?

4. What type of biological sample is best suited for flow cytometric analysis?

5. What is the name given to the portion of the fluid stream where the cells are
located?

6. When cells labeled with fluorescent molecules pass through the focused laser
beam, what two types of light signals are generated?

7. Light emitted from a particle is collected by

_____________________________
8. The electronic signal produced by the detectors is proportional to:

9.All flow cytometric measurements can be made simultaneously on a single cell.

TF

10 Particles must be in single-cell suspension before flow cytometric analysis.

TF