🦠Cell Biology Unit 22 – Techniques in Cell Biology

Key Concepts and Terminology

• Cell biology focuses on the study of the structure, function, and behavior of cells, the fundamental units of life
• Cells are classified into two main types: prokaryotic cells (bacteria and archaea) and eukaryotic cells (animals, plants, fungi, and protists)
• Organelles are specialized structures within eukaryotic cells that perform specific functions, such as the nucleus, mitochondria, endoplasmic reticulum, and Golgi apparatus
• The cell membrane, composed of a phospholipid bilayer, selectively controls the movement of substances in and out of the cell
• The cytoskeleton, consisting of microfilaments, intermediate filaments, and microtubules, provides structural support and enables cell movement
• Cell signaling involves the communication between cells through chemical messengers, such as hormones, neurotransmitters, and growth factors
• The cell cycle is the series of events that lead to cell division and replication, consisting of interphase (G1, S, and G2 phases) and mitosis (prophase, metaphase, anaphase, and telophase)
  • Interphase is the longest phase of the cell cycle, during which the cell grows, replicates its DNA, and prepares for division
  • Mitosis is the process of nuclear division, resulting in the formation of two genetically identical daughter cells

Cell Structure and Organization

• Eukaryotic cells are organized into distinct compartments, each with specific functions and structures
• The nucleus contains the cell's genetic material (DNA) and is surrounded by a double-layered nuclear envelope with nuclear pores for selective transport
• The endoplasmic reticulum (ER) is a network of membrane-bound channels and sacs involved in protein and lipid synthesis, modification, and transport
  • The rough ER is studded with ribosomes and is the site of protein synthesis and modification
  • The smooth ER lacks ribosomes and is involved in lipid synthesis, detoxification, and calcium storage
• The Golgi apparatus is a stack of flattened membrane sacs that modifies, packages, and sorts proteins and lipids for transport to their final destinations
• Mitochondria are the powerhouses of the cell, generating ATP through the process of cellular respiration
• Lysosomes are membrane-bound organelles containing digestive enzymes that break down and recycle cellular waste, foreign particles, and damaged organelles
• Peroxisomes are organelles that detoxify harmful substances and break down fatty acids, generating hydrogen peroxide as a byproduct
• The cytosol is the fluid-filled region of the cytoplasm, containing dissolved nutrients, ions, and proteins

Microscopy and Imaging Techniques

• Light microscopy uses visible light and a system of lenses to magnify small specimens, allowing for the observation of cell structure and morphology
• Bright-field microscopy is the most basic form of light microscopy, in which the specimen appears dark against a bright background
• Phase-contrast microscopy enhances the contrast of transparent specimens by converting phase shifts in light passing through the sample into brightness differences
• Differential interference contrast (DIC) microscopy uses polarized light to create a 3D-like image of the specimen, highlighting surface features and internal structures
• Fluorescence microscopy uses fluorescent dyes or proteins (fluorophores) to label specific molecules or structures within a cell, allowing for their visualization and localization
  • Confocal microscopy is a type of fluorescence microscopy that uses a pinhole to eliminate out-of-focus light, resulting in high-resolution optical sections of the specimen
  • Super-resolution microscopy techniques, such as STED and STORM, overcome the diffraction limit of light to achieve resolutions below 200 nm
• Electron microscopy uses a beam of electrons to create high-resolution images of cell structures, providing detailed information about their ultrastructure
  • Transmission electron microscopy (TEM) passes electrons through thin sections of the specimen, creating 2D images of internal structures
  • Scanning electron microscopy (SEM) scans the surface of the specimen with a focused electron beam, generating detailed 3D images of surface topography

Cell Culture and Maintenance

• Cell culture involves the growth and maintenance of cells in a controlled, artificial environment outside their natural context
• Primary cell cultures are derived directly from living tissues and have a limited lifespan, typically maintaining their original characteristics
• Immortalized cell lines are genetically modified to overcome senescence and can be cultured indefinitely, but may have altered properties compared to their original source
• Culture media provide the necessary nutrients, growth factors, and pH buffering for cell growth and survival
  • Serum, such as fetal bovine serum (FBS), is often added to media as a source of growth factors, hormones, and attachment factors
  • Antibiotics, such as penicillin and streptomycin, are commonly used to prevent bacterial contamination in cell cultures
• Incubators maintain optimal temperature, humidity, and CO2 levels for cell growth, typically set at 37°C, 95% humidity, and 5% CO2 for mammalian cells
• Subculturing, or passaging, involves transferring a portion of the cell population to a new culture vessel with fresh medium to maintain cell growth and prevent overcrowding
• Cryopreservation allows for the long-term storage of cells by freezing them in a protective medium (usually containing DMSO) and storing them in liquid nitrogen
• Cell viability assays, such as trypan blue exclusion or MTT assays, are used to assess the health and survival of cultured cells

Protein Analysis Methods

• Western blotting (immunoblotting) is a technique used to detect and quantify specific proteins in a sample using antibodies
  • Proteins are separated by size using SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and then transferred to a membrane for antibody probing
  • Primary antibodies specifically bind to the target protein, while secondary antibodies conjugated to enzymes (HRP or AP) or fluorophores enable detection
• Enzyme-linked immunosorbent assay (ELISA) is a plate-based assay used to detect and quantify antigens or antibodies in a sample
  • Sandwich ELISA involves capturing the antigen between two antibodies, while competitive ELISA measures the competition between the antigen and a labeled antigen for antibody binding
• Co-immunoprecipitation (Co-IP) is used to study protein-protein interactions by using an antibody to precipitate a target protein along with its interacting partners
• Mass spectrometry (MS) is a powerful tool for identifying and quantifying proteins in complex mixtures based on their mass-to-charge ratios
  • Tandem mass spectrometry (MS/MS) involves multiple rounds of mass spectrometry to generate peptide sequence information for protein identification
• Protein crystallography determines the three-dimensional structure of proteins by analyzing the diffraction patterns of X-rays passed through a crystallized protein sample
• Circular dichroism (CD) spectroscopy measures the differential absorption of left- and right-handed circularly polarized light by proteins, providing information about their secondary structure

Nucleic Acid Techniques

• Polymerase chain reaction (PCR) is a method for amplifying specific DNA sequences by using primers, DNA polymerase, and thermal cycling
  • Quantitative PCR (qPCR) or real-time PCR allows for the quantification of DNA or RNA by measuring the accumulation of fluorescent signal during amplification
  • Reverse transcription PCR (RT-PCR) is used to detect and quantify RNA by first converting it to complementary DNA (cDNA) using reverse transcriptase
• DNA sequencing determines the precise order of nucleotides in a DNA molecule, with common methods including Sanger sequencing and next-generation sequencing (NGS) technologies
• Gel electrophoresis separates DNA or RNA fragments based on their size and charge by applying an electric field to a gel matrix (agarose or polyacrylamide)
• Southern blotting is used to detect specific DNA sequences in a sample by transferring separated DNA fragments to a membrane and hybridizing with a labeled probe
• Northern blotting is similar to Southern blotting but is used to detect and quantify RNA sequences
• In situ hybridization (ISH) uses labeled complementary DNA or RNA probes to detect and localize specific nucleic acid sequences within intact cells or tissues
• RNA interference (RNAi) is a technique for silencing gene expression by using small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) to target and degrade specific mRNA molecules
• CRISPR-Cas9 is a powerful genome editing tool that uses a guide RNA to direct the Cas9 endonuclease to make targeted double-strand breaks in DNA for precise gene modification or deletion

Cell Fractionation and Organelle Isolation

• Cell fractionation is the process of separating cellular components based on their physical properties, such as size, density, or charge
• Homogenization is the first step in cell fractionation, involving the disruption of cells using mechanical (e.g., Dounce homogenizer) or chemical (e.g., detergents) methods
• Differential centrifugation separates organelles and cellular components based on their size and density by applying increasing centrifugal force
  • Low-speed centrifugation (500-1,000 × g) sediments larger components, such as nuclei and intact cells
  • Medium-speed centrifugation (10,000-20,000 × g) sediments smaller organelles, such as mitochondria and lysosomes
  • High-speed centrifugation (100,000-150,000 × g) sediments microsomes (ER and Golgi fragments) and ribosomes
• Density gradient centrifugation separates organelles based on their buoyant density using a gradient medium (e.g., sucrose or Percoll) and ultracentrifugation
• Affinity purification isolates specific organelles or protein complexes using antibodies or ligands coupled to a solid support (e.g., beads) for selective binding and elution
• Isolated organelles can be further analyzed using biochemical assays, proteomics, or microscopy to study their composition, function, and interactions

Specialized Cell Biology Techniques

• Flow cytometry is a technique for analyzing and sorting individual cells based on their fluorescent and light scattering properties as they pass through a laser beam in a fluid stream
  • Fluorescence-activated cell sorting (FACS) uses flow cytometry to separate and collect specific cell populations based on their fluorescent labeling
• Microinjection is a method for introducing substances (e.g., DNA, RNA, proteins, or small molecules) directly into individual cells using a fine glass needle
• Patch-clamp technique is used to study the electrical properties of ion channels in cell membranes by measuring the current flow through individual channels
• Live-cell imaging allows for the real-time observation of dynamic cellular processes using time-lapse microscopy and fluorescent probes or biosensors
• Optogenetics uses light-sensitive proteins (e.g., channelrhodopsin) to control the activity of specific cells or cellular processes with precise spatial and temporal resolution
• Single-cell sequencing captures the genetic information of individual cells, revealing cellular heterogeneity and enabling the study of rare cell types or states
• Tissue clearing methods, such as CLARITY and iDISCO, render tissues transparent by removing lipids and allowing for deep imaging and 3D reconstruction of cellular structures
• Organoids are 3D cell culture models that recapitulate the structure and function of mini-organs, providing a powerful tool for studying tissue development, disease modeling, and drug screening