Flow Cytometry Overview

Flow Cytometry

• Flow cytometry is a technique that measures properties of cells
• A biological sample is labeled with a fluorescent marker
• Cells move through a focused laser beam
• Fluorescent molecules emit light, filtered by detectors (photomultiplier tubes)
• Analog fluorescent signals are converted to digital signals

Flow Cytometer Components

• Flow system (fluidics): Cells in suspension are forced into a single file
• Optical system (light sensing): Focused lasers scatter light and emit fluorescence (filtered and collected)
• Electronic system (signal processing): Emitted light is converted to digitized values, stored in a file for analysis

Flow Cytometry or Fluorescence-Activated Cell Sorter (FACS)

• Sample enters a stream of sheath fluid
• Cells are forced to the center of the stream, forming a single file
• This is done by hydrodynamic focusing
• Only one cell/particle passes through the laser beam at any given moment

Light Scattering

• When light intersects a laser beam ('interrogation point'), light scattering and fluorescence occur
• Light is scattered in all directions
• Scattered light travels to a detector

Forward Scatter Channel (FSC)

• Light scattered in the forward direction (same axis as the laser)
• Intensity of this signal is attributed to cell size, refractive index (membrane permeability)

Side Scatter Channel (SSC)

• Laser light scattered at 90 degrees to the laser path
• Intensity is proportional to cytosolic structure (granules, inclusions)

FSC/SSC Study

• Analyzing FSC and SSC allows cell differentiation (e.g., lymphocytes, granulocytes, monocytes)

Applications of Flow Cytometry

• Quantitative and qualitative analysis of cell populations from various tissues/body fluids (fresh, unfixed)
• Measured properties include particle size, granularity or complexity, fluorescence intensity
• Most common materials analyzed are blood, bone marrow aspirate, and lymph node suspensions

Antibodies in Immuno-Detection

• Antibodies are used in immuno-blotting (western blotting), immunofluorescence, and screening DNA libraries

Western Blotting

• A widely-accepted technique in molecular biology
• Detects specific proteins in a given sample
• Used to detect HIV

Western Blotting Procedure

• Cell supernatant is transferred; cell debris is discarded
• Loading buffer is added to the wells
• Proteins are loaded into wells and gel electrophoresis is run
• Transfer buffer is prepared for transfer tank and gel/membrane equilibration
• The gel, membrane, and filter papers are assembled
• The gel/membrane/filter paper sandwich is placed in the transfer tank which is filled with transfer buffer
• The transfer tank is connected to the power supply
• Transfer is initiated

Immunofluorescence

• Tissue containing the antigen of interest
• Monoclonal antibody labeled with fluorochrome is added
• Unbound antibodies are removed by washing
• Examination by fluorescence microscopy
• Used for detection of Toxoplasma

Immuno-fluorescence (indirect)

• Known antigen is present
• Patient serum is added
• Labeled anti-human globulin antibody is added
• Examination by fluorescence microscopy
  • Detection of Leishmania

DNA Library Screening

• Identifying specific colonies containing plasmids of interest
• Radioactive labeled DNA probe is used
• DNA is bound to paper
• Identification of specific colonies using autoradiography
• Lysing and denaturing of bacteria occurs

Haptens

• Molecules with a low molecular weight.
• Not immunogenic on their own
• Can bind to immune cells, but don't induce immune response
• Require coupling to a carrier protein to induce an immune response

Haptens and Immunogenicity

• Haptens can induce an immune response when covalently bound to a carrier protein (hapten-carrier conjugate)
• Carrier proteins are antigenic and immunogenic(generate immune response)
• Peptides from carrier protein bind to class II MHC and stimulate helper T cells
• Helper T cells then produce interleukins stimulating B cells

Karl Landsteiner

• Discovered blood typing in 1901, earning a Nobel Prize
• Classified human blood into the A-B-O system
• Developed an important technique involving immunizations of rabbits and haptens-carrier conjugates, enabling more specific antibody reactions
• Showed the specificity of antibodies via cross-reactivity
  • Antibody response is specific to the antigen it is elicited by (hapten)

Vaccine Design and Production

• Vaccines are biological preparations that establish or improve immunity to a disease.
• Vaccines typically use killed or live (non-virulent) microbes
• The body produces antibodies to the microbe but doesn't develop the disease
• If exposed again, a ready supply of antibodies will defend against the disease
• Production of vaccine proteins in E. coli and other methods

Vaccine Problems

• Side effects (allergic reactions, aches, pains, fever)
• Consistent virus-based vaccine production (consistent)
• Developing vaccinations for some deadly diseases, such as HIV/AIDS or malaria is risky

Vaccine Production

• Usually, only a few proteins on the surface of the pathogen trigger antibody production
• Biotechnology techniques isolate genes for those proteins, cloning and large production using bacteria like E.coli
• Vaccines are injected, eliciting antibody production. Technologies allow vaccines against Hepatitis B and Meningitis

DNA Vaccines

• Injecting naked DNA into muscles/skin cells elicits an immune response
• DNA alone may not elicit sufficient response for vaccination

Vaccine Delivery Systems

• Production of vaccines (live viruses, coat proteins, or DNA pieces) often requires costly facilities and procedures