Flow Cytometry Basics

Flow Cytometry

• Flow cytometry is used to analyze biological samples.
• Samples are labeled with fluorescent markers.
• Cells flow in a single file through a laser beam.
• Fluorescent molecules are activated and emit light.
• Light is filtered and detected by detectors (photomultiplier tube).
• Analog fluorescent signals are converted to digital signals.

Flow Cytometer Components

• Flow system (fluidics): Cells are suspended and passed in a single file.
• Optical system (light sensing): Laser beam scatters light and emits fluorescence to measure size, granularity, and fluorescence intensity.
• Electronic system (signal processing): Emitted light is converted to digital signals for analysis.

Forward Scatter Channel (FSC)

• Light scattered in the forward direction is measured.
• Intensity is related to cell size and refractive index.

Side Scatter Channel (SSC)

• Light scattered at 90 degrees to the laser path is measured.
• Intensity is proportional to the amount of cytosolic structures (granules, inclusions) in the cell.

Study of FSC and SSC

• Allows for differentiation of different cell types.
• Allows identification of lymphocytes, red blood cells, debris, dead cells, granulocytes, and monocytes.

Method Application

• Qualitative and quantitative analysis of cell populations.
• Uses fresh, unfixed tissue or body fluid.
• Measured properties include particle size, granularity, internal complexity, and fluorescence intensity.
• Common materials for analysis: blood, bone marrow aspirate, and lymph node suspensions.

Antibodies in Immuno-Detection Methods

• Immuno blotting (Western blot)
• Immunofluorescence
• Screening of DNA libraries

Western Blot

• A widely accepted technique for detecting specific proteins.
• Used to detect proteins like HIV.
• A set up is used with filter paper, gel, and membrane.
• Protein samples are run through gel electrophoresis.

Immunofluorescence

• Tissue containing the antigen of interest is identified.
• Monoclonal antibodies are added, labeled with a fluorochrome.
• Unbound antibodies are removed.
• Examined under a fluorescence microscope to detect the antigen.
• Used for detecting Toxoplasma and Leishmania.

Screening DNA Libraries

• Radioactively labeled DNA probe is used to identify colonies of bacteria with recombinant plasmids.
• DNA from the colonies is isolated and analyzed.
• The position of colonies with plasmids of interest is determined through autoradiography.

Haptens

• Low molecular weight molecules.
• Small organic molecules that are antigenic but not immunogenic.
• Bind to immune cells but do not induce a humoral or cell-mediated response.

Why Haptens are Not Immunogenic

• By themselves, they cannot function as immunogenic epitopes.
• Multiple hapten molecules coupled to a carrier protein or non-immunogenic homopolymer make them accessible to the immune system, allowing them to function as immunogens.

Hapten-Carrier Conjugates

• Haptens induce an immune response when covalently bound to a larger protein called a carrier protein.
• Carrier proteins are antigenic and immunogenic.

Karl Landsteiner

• 1901: Discovered the A-B-O blood typing system.
• Awarded the Nobel Prize twenty-nine years later.
• Demonstrated that human blood types and properties are not the same.
• Created the key for matching blood types from a donor to a patient.
• Mixing a wrong type could result in problematic and sometimes fatal consequences.

Vaccine

• Biological preparation establishing or improving immunity to a particular disease.
• Edward Jenner, 1796, observed protection against smallpox using cowpox.

Vaccine Design and Production

• Vaccines use either killed or live microbes.
• Vaccination introduces a non-virulent microbe.
• Your body produces antibodies against the microbes, but you avoid the disease.
• Subsequent exposure to that microbe results in a ready antibody supply for defense.

Problems with Vaccines

• Can cause allergic reactions, aches, or pains, and fever.
• Consistent production of certain virus-based vaccines is difficult.
• Vaccination against diseases like HIV/AIDS and malaria is complicated.

Vaccine Production

• Typically, pathogen proteins trigger antibodies.
• Isolate the pathogen gene for antibody production and insert into bacteria like E. coli for large-scale protein production.
• Using biotechnology, scientists developed vaccines against Hepatitis B and Meningitis.

DNA Vaccines

• Injecting naked DNA into muscle or skin cells triggers an immune response.
• Researchers have been trying to produce DNA-based vaccines, but they still have limitations.

Vaccine Delivery Systems

• Current vaccine development often uses live viruses, coat proteins or DNA-based methods.
• Large-scale vaccine production requires costly facilities and procedures.