Tools in Cell Biology

Illuminating the Cell: Essential Tools in Cell Biology

Dr. Guilherme Costa
Wellcome-Wolfson Institute for Experimental Medicine
Email: g.costa@qub.ac.uk
Course: BMS2110, Theory 2
Date: 17th September 2025

Importance of Tools in Cell Biology

Fundamental Unit of Life

  • Cells are regarded as the basic structural and functional units of all living organisms.

  • Understanding cellular mechanisms is crucial for explaining health states and diseases.

Types of Organisms

  • Unicellular organisms: Composed of a single cell (e.g., bacteria, yeast).

  • Multicellular organisms: Composed of numerous cells organized into tissues and organs.

Biological Organization

  • Organism

    • Organ System

    • Tissue

      • Cell

  • Analogy: Cells are like bricks in a wall, forming the foundational structure of biological systems.

Role of Specialized Tools in Cell Biology

Visualization and Measurement

  • The study of cell biology heavily depends on specialized tools that can:

    • Visualize cellular components and processes.

    • Manipulate cellular functions.

    • Measure various cellular activities.

Key Cellular Processes

  1. Cell Division

  2. Cell Migration

  3. Cell Death

Technological Innovations Driving Progress

Key Examples of Innovations

  • Microscopy: A range of techniques used to magnify small structures.

  • Cell Culture: The process of growing cells in controlled environments.

  • Types of Microscopy Technologies:

    • Confocal Microscopy

    • Super Resolution Microscopy

Cellular Structures Analyzed

  • Trafficking of vesicles

  • Tubulin arrangement in microtubules

  • Different types of cell cultures (2D & 3D)

  • Neural progenitor cells and mature neurons

Learning Outcomes

At the conclusion of this session, participants will be able to:

  • Describe key techniques employed in studying cells and their functional dynamics.

  • Understand the underlying principles of cell culture, microscopy, and molecular tools used in research.

  • Recognize applications of these scientific tools in biomedical research settings.

Tools in Cell Biology

Cell Culture

  • Definition: A simplified model system used for biological research.

  • Benefits:

    • Facilitates the investigation of specific research questions.

    • Enables genetic manipulation at the gene level.

Types of Cells Used in Culture

  • Neurons

  • Fibroblasts

  • Osteoblasts

  • Chondrocytes

  • Melanocytes

  • Endothelial cells

  • Myotubes (muscle cells)

Importance of Cell Culture

Culture Setup Requirements

  • 2D Cultures: Utilized on various substrates such as:

    • Culture media

    • Dishes

    • Plates

    • Wells

  • Environmental Conditions:

    • Maintained at 37°C with a CO2 concentration of 5%.

  • Nutrient-Rich Medium: Ensures proper growth and maintenance of cell viability.

Physiological Models Generated

  • Models of various organs and systems including:

    • Muscle, liver, bone marrow, kidney, and brain.

    • Lungs, heart, and vasculature modeling.

Advanced Culture Models

Types of Advanced Models

  1. 3D Cultures:

    • Organoids: Minimized models of organs, composed of multiple cell types.

    • Microphysiological systems (MPS): Mimics multi-organ physiology.

    • Features interconnected systems, modeling physiological interactions in organs such as the lungs and heart.

Components of Culture Systems

  • Must include extracellular matrix (ECM), cell types, and associated cell-cell interactions.

  • Media circulation through microfluidic systems enhances experimental accuracy.

Lifespan of Cells in Culture

Primary Cells

  • Characteristics:

    • Limited lifespan and eventually stop dividing or die.

    • Typically obtained directly from tissues (e.g., primary cell cultures).

Immortalized Cell Lines

  • Definition: Cell lines that can be propagated indefinitely under appropriate conditions.

    • Can be derived from primary cells.

    • May be naturally immortal (e.g., cancer cells).

  • Cell Lines: Capable of repeated propagation without significant loss of viability.

Tools in Cell Biology

Microscopy

Types of Microscopy Techniques

  1. Electron Microscopy:

    • Utilizes beams of electrons instead of light, resulting in considerably higher resolution images.

    • Applications include visualizing lymphocytes or cancer cells in metastasis.

  2. Scanning Probe Microscopy:

    • Measures surface characteristics at the atomic scale using a tiny probe.

    • Capable of detecting specific cellular structures such as potassium channels in lipid membranes.

  3. Optical Microscopy:

    • The most widely used microscopy method employing visible light.

    • Uses lenses or mirrors to reflect light for image magnification.

    • Includes techniques like transmitted light and fluorescence microscopy, with focus on neuronal cell cultures.

Molecular Tools

Protein Detection Techniques

  1. Western Blotting:

    • Employs antibodies to detect specific proteins post-gel electrophoresis.

    • Provides insights into:

      • Protein size

      • Relative abundance in different samples

      • Post-translational modifications of proteins.

Basic Steps of Western Blotting:

  1. Sample Preparation:

    • Preparing the sample lysate and adding sample buffer.

  2. Gel Electrophoresis:

    • Migration of proteins based on size through the gel matrix (high and low molecular weight).

  3. Membrane Transfer:

    • Transferring proteins separated in gel onto a membrane.

  4. Detection:

    • Utilizing HRP-conjugated secondary antibody and chemiluminescent substrates for signal detection.

Examples of Western Blotting

  • Analysis of myoblasts differentiating into myotubes, highlighting day-dependent changes in protein expression:

    • Cadherin-2 (98 kDa)

    • Beta-Sarcoglycan (43 kDa)

    • Aquaporin-1 (38 kDa)

    • Topoisomerase-I (20 kDa)

Immunochemistry

  • Utilizes antibodies for protein detection in tissue samples (e.g., pancreatic islet cells).

  • Visual representation: protein iNOS marked brown through a colored substrate reaction.

Immunofluorescence Techniques

  • Immunofluorescence Assay: Used to detect proteins tagged with fluorophores specified by primary and secondary antibodies.

  • Example: Glucagon protein marked in red on pancreatic islet cells.

Loss-of-Function (LoF) Approaches

Objective of LoF Approaches

  • Aim to reduce or ablate gene function to elucidate its biological role.

  • Commonly targets genetic material: DNA or RNA.

Common LoF Approaches

  1. CRISPR:

    • Targets DNA to result in permanent modifications.

    • Utilizes guide RNA (gRNA) and Cas9 enzyme.

    • High specificity, used to study essential genes.

  2. siRNA (small interfering RNA):

    • Targets mRNA for transient silencing of gene expression.

    • Less specificity, beneficial for studying variable gene functions.

Agarose Gel Electrophoresis

Definition and Purpose

  • Technique employed to separate biological molecules such as nucleic acids or proteins based on size.

  • An electric current pushes molecules through an agarose gel matrix:

    • Smaller molecules migrate faster compared to larger ones.

Validation of LoF Using Agarose Gel Electrophoresis

  • Identifying mutational changes in CRISPR lines and analyzing siRNA-induced knockdowns in target genes.

Practical Sessions
Weekly Schedule

  1. Cell Culture Practical

    • Date: Tue, 23rd Sept 10:00; Group 1-6

    • Date: Thu, 25th Sept 14:00; Group 7-12

  2. Immunofluorescence Practical

    • Date: Tue, 4th Nov 09:30; Group 13-24

    • Date: Tue, 4th Nov 14:00; Group 1-12

  3. Agarose Gel Electrophoresis Practical

    • Date: Tue, 25th Nov 10:00; Groups 1-12

    • Date: Tue, 25th Nov 14:00; Groups 13-24

Learning Objectives

  • Understanding core principles and applications of techniques practiced above in analyzing cellular functions and behaviors.

Case Study: RNA Binding Protein IGF2BP1 in Angiogenesis

Overview

  • Concept: Angiogenesis refers to the formation of new blood vessels from existing ones.

  • Specific Protein Studied: IGF2BP1, an RNA-binding protein implicated in this biological process.

Experimental Procedures

  1. Targeting IGF2BP1:

    • Cell Type: Endothelial cells.

    • Method of Knockdown: Using siRNA targeting IGF2BP1.

    • Collection of lysates for assessing protein expression.

  2. Analysis: Examination of changes in endothelial cell movement and sprouting abilities post-knockdown treatment.

Results from Experiments

  • Observations include:

    • Downregulation of IGF2BP1 via siRNA treatment reduces sprout lengths in angiogenic assays.

    • Techniques utilized include microscopy, immunofluorescence, and measurement of cell displacement and speed under experimental conditions.

Summary of Learning Outcomes

  • Participants can now:

    • Describe key experimental techniques utilized to investigate cell biology.

    • Comprehend the underpinning principles of cell culture, microscopy, and molecular biology tools.

    • Recognize the significance of these tools in advancing biomedical research.

Additional Information and Thank You

  • For further questions or feedback, contact:

    • Dr. Guilherme Costa
      Email: g.costa@qub.ac.uk

  • Student feedback portal available at: https://forms.office.com/e/kbtcD3uvHM