Flow Cytometry Overview

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Questions and Answers

What is one way haptens can be utilized in medicine?

They prevent drug allergies

They cure diseases

They act as vaccines

They are used in pregnancy tests (correct)

What role do proteins on the surface of pathogens play in vaccine production?

They are the primary cause of diseases

They prevent vaccine side effects

They trigger antibody production (correct)

They cause serious allergic reactions

What is a potential issue related to the development of vaccines?

Vaccines can only be made from live pathogens

All vaccines are guaranteed to be effective

Vaccines always cause severe side effects

Consistent production of virus-based vaccines is challenging (correct)

What method can be used to produce large quantities of vaccine proteins?

Isolating the gene and inserting it into E. coli Signup and view all the answers

Which of the following drugs is known to potentially elicit an allergic reaction?

Aspirin Signup and view all the answers

What is a significant advantage of vaccines?

They help improve immunity without causing disease Signup and view all the answers

What is a common side effect of vaccines?

Allergic reactions Signup and view all the answers

How did the term 'vaccine' originate?

From Edward Jenner's use of cow pox Signup and view all the answers

What is the primary purpose of a western blot in molecular biology?

To detect specific proteins in a sample Signup and view all the answers

Why are haptens considered non-immunogenic on their own?

They cannot induce an immune response by themselves. Signup and view all the answers

What role does the carrier protein play in the immunogenic response involving haptens?

It presents the hapten to helper T cells. Signup and view all the answers

In what process are antibodies most likely utilized?

Immunofluorescence Signup and view all the answers

What is a key feature of the hapten-carrier conjugate?

It helps haptens to be recognized by the immune system. Signup and view all the answers

What analytical technique is widely accepted for detecting specific proteins?

Western blot Signup and view all the answers

What do activated helper T cells produce after recognizing the hapten-carrier complex?

Interleukins Signup and view all the answers

Which of the following methods is NOT listed as an antibody use in immuno-detection?

Flow cytometry Signup and view all the answers

Study Notes

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

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Description

This quiz covers the principles of flow cytometry, including its components and how it functions to analyze cell properties. Learn about the optical and electronic systems that help convert fluorescent signals to digital data. Explore the technique's applications in cell sorting and analysis.