BIOL 4426: CELLULAR PHYSIOLOGY - ELECTRONIC LECTURE NOTES - PART II. METHODS FOR THE STUDY OF EUKARYOTIC CELLS

50 vocabulary flashcards covering key methods for the study of eukaryotic cells, including microscopy, cell purification, cell culture, subcellular fractionation, protein analysis, and recombinant DNA technologies, based on the provided lecture notes.

Brightfield Microscopy

Microscopy using visible light where light goes through the object, resulting in a lighter field with a darker sample.

Resolution (Microscopy)

The smallest distance between two points that can be distinguished as separate; typically ~0.2 µm for brightfield microscopes.

Fixation (Microscopy)

A chemical process used to preserve cellular structure, which typically kills the cells.

Histological Staining

Techniques using dyes to stain cellular structures for better visualization.

Hematoxylin-Eosin (H&E)

The most common histological staining procedure, using two dyes.

Histochemical Staining

A staining technique designed to detect specific enzymes within cells or tissues.

Immunohistochemical Staining (IHC)

A staining technique used to detect specific proteins using an antibody conjugated with an enzyme.

Microtome

An instrument used for sectioning tissue, typically by embedding it in resin and cutting with a knife, before microscopic observation.

Fluorescence Microscopy

Microscopy that uses fluorescent dyes (fluorophores) that absorb light at one wavelength and emit it at a different, longer wavelength.

Fluorophores

Fluorescent dyes that absorb light at one wavelength (color 1) and emit light at a different (longer) wavelength (color 2).

Immunofluorescence Microscopy (IF)

A technique to detect a target antigen using a labeled fluorescent antibody, known for its sensitivity and good resolution.

Confocal Microscopy

A type of fluorescence microscopy that uses a laser beam focused on a very thin plane to produce sharper images, even with relatively thick samples.

Deconvolution Microscopy

A fluorescence microscopy technique that mathematically analyzes several images taken at different focal planes to improve sharpness.

Dynamic Fluorescence Microscopy

Techniques used to detect molecules in living cells, such as fluorescent detection of Ca2+ ions, GFP fusion proteins, or FRET.

FRET (Förster Resonance Energy Transfer)

A technique used in dynamic fluorescence microscopy to visualize protein-protein interactions.

Phase Contrast & Nomarsky Interference (DIC) Microscopy

Microscopes that provide high contrast for living unstained samples by using refraction and diffraction of light.

Transmission Electron Microscopy (TEM)

A microscopy technique that uses an electron beam (instead of light) to achieve very high resolutions (<0.1nm) for internal cellular structures.

Ultramicrotome

A specialized microtome used to produce the very thin sections required for Transmission Electron Microscopy (TEM).

Immunogold

A technique used in electron microscopy where an antibody is attached to a gold bead to specifically stain and detect cellular components.

Scanning Electron Microscopy (SEM)

A microscopy technique that uses a narrow electron beam to scan the surface of a sample, causing secondary electrons to bounce off, providing very high-resolution surface images.

Tissue Disaggregation

A methodology involving the use of enzymes (especially proteases) to remove proteins holding cells together in a tissue, breaking it down into individual cells.

Trypsin

A commonly used protease in cell biology to separate cells from tissues by attacking extracellular matrix and cell adhesion molecules.

EDTA

A Ca2+ ion chelator frequently used in cell disaggregation because removing calcium weakens cell adhesion.

Centrifugation (Cell Separation)

A method to separate cells based on differences in their physical properties, such as size or density.

Fluorescence Activated Cell Sorter (FACS)

An advanced instrument used for flow cytometry and cell sorting, capable of detecting fluorescently labeled cells and separating them based on fluorescence level, size, and shape.

Primary Cell Cultures

Cell cultures obtained directly from tissues, which are genetically normal and often retain characteristics similar to the original tissue, but have limited division capacity.

Fibroblasts

A type of cell that is generally easy to culture in primary cell cultures because they produce their own extracellular matrix and are not highly delicate regarding growth factor requirements.

Crisis Period (Cell Culture)

A stage in primary cell cultures where cells stop dividing and eventually die by senescence, typically after 45-50 cell divisions.

Senescence (Cell Culture)

The process of cellular aging in primary cell cultures where cells lose their ability to divide, often linked to telomere length reduction.

Immortalized Cell Line

A cell line derived from primary cultures (often rodent) where a few cells spontaneously survive the crisis period and gain the ability to divide indefinitely.

Oncogenically Transformed Cell Lines

Cell lines, often derived from human tumors, that have acquired genetic alterations allowing them to divide indefinitely, such as the HeLa cell line.

HeLa Cell Line

The first human immortalized cell line established in 1952 from a uterine carcinoma, historically significant for its widespread use in biomedical research.

Cell Homogenates

Preparations of cellular material where the plasma membrane has been disrupted to release organelles, aiming to keep the organelles active and intact.

French Press

A mechanical homogenization technique that disrupts cells by forcing the sample through a narrow hole, causing a significant pressure drop.

Dounce Homogenizer

A manual glass homogenizer where cells are lysed by forcing them through a narrow space between a glass tube and a pestle.

Differential Centrifugation

A stepwise centrifugation technique at increasing speeds used to separate organelles based on their size, yielding crude subcellular fractions.

Equilibrium Density Gradient Centrifugation

A purification method where a sample is centrifuged in a density gradient until organelles separate into distinct bands based on their specific densities.

Affinity Purification (Organelles)

A method to purify specific organelles or vesicles using antibodies that bind to their surface, often coupled to large protein-A beads.

SDS Gel Electrophoresis

A widely used gel electrophoresis procedure that separates proteins primarily by their size, typically after denaturing them with SDS.

IEF (Isoelectric Focusing)

A gel electrophoresis technique that separates proteins based on their isoelectric point (pI), their unique pH at which their net electrical charge is zero.

Isoelectric Point (pI)

The specific pH value at which a protein carries no net electrical charge and will not migrate in an electric field.

2D Gel Electrophoresis

A high-resolution technique that combines Isoelectric Focusing (IEF) in the first dimension and SDS-PAGE in the second dimension to separate thousands of proteins.

Chromatography (Protein Purification)

A purification technique that separates active proteins based on properties like mass, charge, or specific binding, and can be scaled up.

Western Blotting (= Immunoblotting)

A detection method where proteins from a gel are transferred to a membrane and then specific proteins are detected using primary and labeled secondary antibodies.

Mass Spectrometry (Proteins)

An analytical technique that precisely determines the mass of ionized proteins and peptides, used for protein identification and sequencing.

X-ray Crystallography

A technique that determines the three-dimensional structure of proteins by analyzing the diffraction pattern generated when X-rays pass through a protein crystal.

Plasmids

Small, self-replicating circles of DNA commonly used as vectors in DNA cloning, containing an origin of replication, an antibiotic resistance gene, and a multicloning site.

Restriction Enzymes

Enzymes that cut DNA molecules at very specific nucleotide sequences, often leaving 'sticky ends' that facilitate DNA ligation.

Recombinant Plasmids

Plasmids that have been engineered to contain a foreign DNA fragment introduced via restriction enzyme digestion and ligation by DNA ligases.

CRISPR-CAS

A powerful and efficient molecular tool used for endogenous gene modification, allowing precise editing of genomes at specific locations.