BIO3553 Biologie Cellulaire Final

Indirect Immunocytochemistry Technical Aspects

• use of primary antibodies to bind specific targets

• labelled secondary antibodies amplify the signal

• the choice of antibody and labelling method is crucial for detection effectiveness

Confocal Microscopy Technical Aspects

• allows for taking “optical selections” at different depths to create a 3D image

• uses lasers as the light source

• detection of fluorescence emitted by the specimen

• the confocal aperture design collects only light focused at the focal point

Immunocytochemistry

• to detect specific molecules (e.g. proteins, RNA)

Confocal Microscopy

• to observe complex 3D structures (cytoskeleton, organelles, chromosomes)

• ideal for observing living cells or subcellular structures

Electron Microscopy

• Scanning Electron Microscope (SEM) = observes the surface of the specimen in 3D

• Transmission Electron Microscope (TEM) = observes internal structures with high resolution

Confocal Microscope Operating Principles

• uses lasers and confocal apertures to collect only the light emitted from the focused point

• generates 2D images by scanning through different layers of the specimen

SEM Operating Principles

• uses electrons to observe the surface of the specimen

• electrons bounce off the specimen, creating an image

TEM Operating Principles

• electrons pass through the specimen to create a high-resolution image of internal structures

Choice of Microscopy

• confocal = ideal for 3D structures and fluorescence imaging

• electron = chosen based on the need for ultra-high resolution or surface observation

Specimen Preparation

• immunocytochemistry = fixation and labelling with specific antibodies

• SEM = preserve surface properties, dehydration, and metal coating

• TEM = fixation in chemicals, dehydration, and thin sectioning of the specimen

Cell Culture

• isolating and growing cells under controlled conditions to observe their behaviour

Fluorescent Labelling

• using fluorescent markers to identify specific structures within cells

Genetic Engineering

• manipulating DNA to observe the effects of gene knockdown or over expression

Microdissection

• cutting specific sections of cells or tissues for more targeted analysis

High-Resolution Imaging

• using techniques like electron microscopy to observe cellular details

Optical Microscopy

• used to observe living or fixed cells at low resolution

Fluorescence Microscopy

• allows the study of specific molecules within cells using fluorescent markers

Live-Cell Imaging

• observing dynamic processes in living cells in real time

Optical Microscopes Operating Principles

• use visible light to magnify samples

• rely on refraction of light

Fluorescence Microscope Operating Principles

• employ specific wavelengths of light to excite fluorescent molecules in the sample

• allowing the visualization of specific cell components

Cell Culture

• set of processes used to grow cells that have been isolated from the organism

• discoveries

  • cells to be grown can be obtained easily and in large quantities

  • most cultures contain only one cell type

  • several different cellular activities

  • cells can be differentiated in culture

  • animal cell cultures avoid the use of live animals in several types of experiments

• response to treatments

  • drugs

  • hormones

  • growth factors

Important aspects of cell culture

• to maintain their viability and proliferate, cells must be growing in a medium that contains nutrients, vitamins, hormones, growth factors, co factors, and several unknown substances isolated from lymph, blood serum, embryo homogenate

• cells must grow under specific environmental conditions (temp and CO2) and highly sterile

Types of Cell Culture

• primary = directly from animal and embryo

  • cells are obtained by dissociation in a proteolytic enzyme

  • trypsin = digest extracellular domains of proteins that control cell adhesion

• secondary = already grown cells frozen in small vials, in a liquid nitrogen tank

To avoid senescence and death

• cells are usually grown in suspension or petri dishes and must be regularly subcultured

Differentiating Properties: Animal or Plant Crops

• Fibroblasts = continue to secrete collagen

• Myoblasts = when derived from embryonic skeletal muscles, they fuse and form muscle fibers that contract spontaneously inside a petri dish

• Neural Stem Cells = form axonal extensions electrically excitable and able to form synapses

• Epithelial Stem Cells = form extensive sheets retaining the properties of an intact epithelium

Polyclonal Antibodies

• produced by inoculation of a target protein to animals

• the antibodies are then isolated from the animal’s serum

• the resulting antibodies are a mixture, recognizing multiple antigenic sites of the protein

• only a fraction of these antibodies are specific to the target proteins

Monoclonal Antibodies (Via hybridoma cells)

• hybridoma technology allows for the production of an unlimited supply of identical and highly specific antibodies

• these antibodies provide increased specificity for antibody-based methods

Monoclonal Antibody Method

• objective = to produce a homogeneous population of antibodies from a single B cell clone

• challenge = b cells have short life in culture

• Solution = merge the B cells of an immune mouse with transformed B cell lines to create hybridomas

• result = hybridomas are stable and durable cells that produce a single type of monoclonal antibody, each recognizing a specific antigenic site

Benefits and Uses of Cell Culture

• in cell biology = monoclonal antibodies can be used to

  • locate and track proteins in cells or tissues

  • purify proteins for structural and functional studies

• in disease treatment

  • cancer = monoclonal antibodies can block signal receptors on cancer cells to prevent their proliferation

  • autoimmune diseases (e.g. rheumatoid arthritis) = they can target and inhibit immunostimulating molecules to reduce inflammation

Fractioning of Cell Content by Differential Centrifugation

• method isolates specific cell organelles or components by ultracentrifugation

• process = during centrifugation,microsomes (membrane fragments) and ribosomes are removed from the suspension

• after removal of the ribosome, the supernatant contains soluble cellular material and smaller particles

• to study organelles or enzymes, they are isolated and purified using this technique

Principle of Fractioning

• particles of different sizes and densities deposit at different speeds when subjected to variable centrifuging speeds, allowing the separation of specific organelles or components according to their density

Cell Fractioning with ultracentrifuge

• objective = to separate and isolate specific components of a cell homogenized, often for protein purification

• process = the cell extract is spun at high speed in an ultracentrifuge to separate subcellular components according to their size and density

• protein source = may come from natural tissue cells or recombined cells designed to produce large amounts of a specific protein

Steps for Protein Extraction and Purification

1. cell destruction = methods include osmotic shock, ultrasonic vibration, or mixing

2. organelle protection = the destruction process helps to preserve organelles such as mitochondria, nucleus, golgi apparatus, lysosomes, and peroxisomes

3. homogenized (Extract) = this thick slurry contains various organelles and microsomes (vesicles derived from the ER) which are then separated by differential centrifugation

Separation of Cell Components by Centrifugation

• low speed = larger components (for example, the core) settle first, forming a pellet

• slightly higher speed = mitochondria form a pellet

• very high speed = first, the vesicles settle, follow by the ribosomes, each step separating the components according to size and density

sedimentation rate and eq’m

• sedimentation rate = method refines the separation after centrifugation using a sucrose gradient

• process = homogenized is placed on a saline solution, and during centrifugation, the cellular components move through the solution, forming bands at different levels depending on their size and shape

• a density gradient is created (denser at the bottom), allowing better separation of components which form distinct bands that can be collected, their sedimentation rate being determined by size and shape (sedimentation coefficient, S)

Sedimentation E’qm

• process = ultracentrifugation separates compoenents based on their flotation density rather than size or shape

• the sample is sedimented by a pronounced density gradient (often with sucrose or cesium chloride)

• each compoenent moves along the gradient until it reaches a positions where the ensity of the solution corresponds to its own density, causing it to float and stop moving

• strips = separate strips are formed inside the centrifuge tube, with the densest components at the bottom

Chromatography Technique

• purification = removes contaminants

• characterization = measurement of total protein using total nitrogen (16% dry weight for most proteins)

• liquid chromatography

  • separation = components move through a porous matric

  • phases

    • mobile = moving solvent

    • immobile = matrix that binds proteins for separation

Three types of matrices for chromatography

• ion exchange = separates proteins according to the charge

  • column is filled with beads that carry positive or negative charges, attracting proteins with opposite charges

• hydrophobic = separates proteins by their hydrophobicity

  • column contains beads with hydrophobic regions, which bind proteins with hydrophobic zones

• gel filtration = separates by size

  • smaller proteins enter the pores of the beads and move more slowly, while larger ones bypass the pores and emerge first

• affinity = separates proteins according to their ability to bind to specific small molecules

  • a substrate is covalently attached to the beads

  • proteins that bind to it are retained and can be purified

Polyacrylamide gel electrophoresis

• objective = used to separate proteins according to their load, size, and shape

• process= proteins are loaded into a gel made of acrylamide polymers and exposed to an electric field

• proteins move through the gel = negatively charged proteins migrate to the positive electrode (cathode)

• factors affecting movement = proteins with a higher load migrate faster

  • larger proteins move more slowly

• after electrophoresis = the gel is tinted to visualize proteins

  • the gel can be cut into fractions to isolate proteins

Western blotting

• after gel electrophoresis

• proteins are transferred to a nitrocellulose membrane, retaining their position from the gel

• proteins are identified by specific antibodies

Gel Electrophoresis for nucleic acids

• objective = separates nucleic acids (DNA or RNA) by molecular weight, according to the length of the nucleotide segments

• small molecules = separated by polycrylamide gel electrophoresis

• large molecules = separated by electrophoresis on agarose gel (larger pores for large molecules)

• visualization = after electrophoresis, the gel is socked in an ethidium bromide dye, which intersperes with DNA and fluoresces under UV light

• high sensitivity = The technique allows to distinguish DNA or RNA molecules that differ by a single nucleotide, useful for DNA sequencing. (Caution: wear gloves and protective glasses when handling ethidium bromide.)