Cell Biology Methods
Cell biology methods allow researchers to study cell structure, function, and physiology at the molecular level. These techniques are fundamental to understanding disease mechanisms and developing new therapies.
Microscopy
- Light Microscopy: Resolution limit ~200 nm. Bright field (standard H&E staining), Dark field, Phase contrast (living cells), Polarized light (birefringent materials — urate crystals in gout, amyloid with Congo red).
- Fluorescence Microscopy: Fluorescent dyes or GFP fused to proteins. FISH (Fluorescence In Situ Hybridization): Fluorescent probes hybridize to specific chromosomal regions → detect translocations, gene amplification, chromosomal abnormalities (e.g., HER2 amplification, BCR-ABL).
- Confocal Microscopy: Laser scanning; eliminates out-of-focus light; 3D images of cells and tissues. Live cell imaging.
- Electron Microscopy: - TEM (Transmission EM): Ultra-thin sections; resolution ~0.1 nm; internal cell ultrastructure (organelle morphology, virus particles, mitochondria). - SEM (Scanning EM): Surface topography; 3D surface images of cells. - Used to identify viruses (HIV, coronavirus), glycogen storage diseases, mitochondrial disorders.
- Super-Resolution Microscopy: PALM, STORM, STED — break diffraction limit; resolve structures down to 10–20 nm. Nobel Prize in Chemistry 2014.
Cell Fractionation
Differential centrifugation separates organelles by size and density. Cells disrupted (homogenized) → centrifuge at increasing speeds:
- 600g: Nuclei + cell debris pellet
- 5,000g: Mitochondria, chloroplasts, lysosomes
- 20,000g: Peroxisomes, heavy ER
- 100,000g: Ribosomes, light ER, Golgi vesicles
- Supernatant: Cytosol
Density gradient ultracentrifugation (CsCl or sucrose) separates by buoyant density (e.g., DNA types, lipoprotein types).
Flow Cytometry
Cells in suspension flow single-file through laser beam → multiple parameters measured per cell simultaneously: Forward scatter (cell size), Side scatter (granularity), Fluorescence (surface markers, DNA content, intracellular proteins). Can sort cells (FACS — Fluorescence Activated Cell Sorting). Applications:
- Immunophenotyping: CD4/CD8 T-cell counting (HIV/AIDS monitoring), leukemia/lymphoma classification
- Cell cycle analysis: DNA content (PI stain) → G1, S, G2/M phase fractions
- Apoptosis detection: Annexin V (early apoptosis), PI (late apoptosis/necrosis)
- Intracellular cytokine staining
Cell Culture
- Primary cells: Directly from organism; limited lifespan (Hayflick limit); most physiologically relevant
- Cell lines: Immortalized; easy to culture; may not reflect normal biology. HeLa cells (Henrietta Lacks' cervical cancer — first immortal human cell line).
- Stem cells: iPSCs (induced pluripotent), ESCs — can differentiate into various cell types
- Organoids: 3D mini-organs grown from stem cells; model human diseases
Immunoprecipitation (IP) & Co-IP
Antibody linked to agarose beads captures target protein from cell lysate. Co-IP pulls down interacting proteins simultaneously → study protein-protein interactions. ChIP (Chromatin IP): Crosslink protein to DNA → IP with antibody → analyze DNA sequences bound to protein.
CRISPR Screens
Genome-wide CRISPR knockout libraries screen all genes for effect on a phenotype (growth, drug resistance). Essential gene identification, synthetic lethality screens for cancer drug targets.