Untitled Quiz

Cell Cultures

• Cell cultures are lab techniques isolating and maintaining cells in artificial conditions.
• Cells can be isolated from any organism, but some are harder than others.
• Temperature, pH, etc. are controlled to allow cell growth and maintenance.

Types of Cell Cultures

• Primary Cultures: Cells directly taken from a tissue.
• Cell Lines: Cultures of cells that can divide, sometimes indefinitely.
  • Finite Lines: Divide a limited number of times before senescence.
  • Continuous Lines: Divide indefinitely, often from tumor biopsies or through transformation.
  • Cell Strains: Subpopulations selected from a cell line, typically genetically similar.

Cell Isolation

• Isolation is specific to each cell type, it is a mechanical and enzymatic process
• Isolated cells are placed in culture medium to form a primary culture.
• The culture proliferates until it fills the dish, then a portion is transferred to new medium to form a cell line.

Cell Growth

• Cells are seeded in Petri dishes with simulated in vivo conditions (temperature, pH).
• Initial cell numbers decrease due to isolation damage; growth then accelerates.
• Finite lines reach senescence; continuous lines proliferate indefinitely.

Adherent or Monolayer Culture

• Cells grow attached to a solid/semi-solid substrate.
• Growth occurs in a monolayer.
• Growth is limited by surface area until enzymatic or mechanical dissociation is performed.

Suspension Culture

• Cells suspended within a liquid culture medium
• Used for non-adhesive cells like blood cells
• Does not require enzymatic/mechanical dissociation
• Requires agitation for adequate gas exchange

Cell Morphology

• Fibroblastic: Bipolar or multipolar, elongated, monolayer.
• Epithelial: Polygonal, monolayer.
• Lymphoblastic: Spherical, suspension.

Primary Cultures vs. Cell Lines

• Primary Cultures: High differentiation, in vitro characteristics similar to in vivo, higher biotransformation capacity, and enzymatic activity. Labor-intensive, heterogeneous, and require a new isolation for every experiment.
• Cell Lines: Provide consistent results; greater homogeneity of genotype and phenotype; infinite proliferation (frozen in liquid nitrogen). Lose some in vivo tissue characteristics, like biotransformation enzymes, in comparison to fresh cells.

Cell Culture Uses

• Research: Models for toxicology, drug development, gene therapy, cancer research
• Industry: Synthesizing compounds; cultivating proteins, vaccines; replacing tissues/organs, cellular agriculture;

Cell Culture Sources

• Cell line banks (ATCC, ECACC, DSMZ)
• Collaborations with other scientists

Laboratory Conditions for Working with Cell Cultures

• Biosafety levels (BSL-1 to BSL-4): corresponding to the danger levels
• Appropriate equipment like Laminar Flow hoods for the work involved.

Cell Culture Contamination

• Chemical: Impurities in medium, buffers or serum.
• Biological: Bacteria, yeast, fungi, mycoplasma, and other cell cultures.
• Detection: Observation (color change, growth rate alterations); molecular biology techniques (PCR).

Cytology Techniques

• Imprint: Pressing a glass slide against the tissue.
• Scraping: Scraping the lesion with a glass slide or a scalpel to collect cells.
• Swab: Using a sterile swab to collect cells from mucous membranes.
• Brush: Using a brush to collect cells from mucous membranes.

Aspirative Cytology

• Using a fine needle aspiration (FNA) to collect cells from lesions or organs.

Sample Processing Techniques (Cytology)

• Low Mucus and protein content: Storage time is limited
• High Protein content: Storage is limited to 24-48h
• High mucus content: Storage is limited to 12-24h
• Immediate processing is best if possible

Blood Cytology Processing

• Smears or centrifugation.
• "Buffy Coat" smear (white blood cells and platelets)

Electron Microscopy

• SEM: 3D images of the external surface of the samples
• TEM: 2D images of the internal structure (requires sectioning)

Cytotoxicity Assays

• Methods to evaluate the physical or chemical properties of damage to cell health/metabolism
• Different assays based on mechanisms, specificity, sensitivity, and equipment used.

Exclusion Assays

• These assays measure the response of cells to a toxic reagent by staining dead ones.

LDH Assay

• Monitors irreversible cell death; based on membrane integrity
• FBS has inherent LDH activity; use in assays may need to be reduced/eliminated.

SRB Assay

• Measures protein synthesis inhibition.

NRU Assay

• Uses Neutral Red, which accumulates in viable cells.
• More sensitive to damage by certain pollutants

Fluorometric Assays

• More sensitive than colorimetric assays.

AlamarBlue (AB Assay)

• Measures enzymatic activity; good for monolayers or suspensions.

CFDA-AM Assay

• Measures cell membrane integrity based on the chemical reaction mediated by esterases.

GF-AFC Assay

• Measures protease enzymatic activity (a good cell viability indicator)

Luminescence assays

• Highly sensitive assays, using ATP level as an indicator of cell viability.

Real-Time Viability Assay

• Allows real-time measurement of cell viability/cytotoxicity
• Detects irreversible cell death; based on membrane damage

Genotoxicity

• Assessing damage to DNA that may not be lethal

Comet Assay

• Measures DNA breaks in cells.
• Quantifies DNA migration distance.

Cell Isolation (for Comet Assay)

• Cell cultures: Cells scraped or trypsinized.
• Blood leukocytes: Isolated by centrifugation.
• Tissue cells: Tissue disaggregated to create cell suspension.
• Plant cells (Nuclei release): Cut with a knife.

Cell Embedding for Comet Assay

• Agarose is used to embed the cell suspensions.

Cell Lysis for Comet Assay

• Detergents (like triton X-100) disaggregate cell membranes; separate DNA from proteins, usually overnight at 4 degrees Celsius.

Alkaline Treatment & Electrophoresis (Comet Assay)

• Treatment in high pH to promote DNA strand migration (electrophoresis).

Staining (Comet Assay)

• This phase uses specialized dyes that react to the stained DNA and make it visible via image analysis.

Visual scoring (Comet Assay)

• Classifies comet tails based on size.
• A range from 0 to 4.
• The number of cells in each class generates the comet score.

Computer Assisted Image Analysis

• Computer analysis of the comet images; it scans the pictures and gives a statistical analysis of each comet, it is more objective.
• Semi-automatic (operator selects comets to be analyzed), Automatic method (slide is scanned, and all comets are analyzed automatically).

Immunohistochemistry/Immunocytochemistry

• Techniques: Identifying antigens.
• Advantages: High sensitivity and Specificity.
• Disadvantages: More complex, higher cost.

Antigens

• Antibody recognition molecules.

Antibodies

• Proteins produced by activated B lymphocytes to recognize antigens.
• Monoclonal: From a single clone of plasma cells, reacting to a single epitope.
• Polyclonal: From multiple lymphocytes, reacting to multiple epitopes.

Immunofluorescence (IF)

• Uses fluorescent dyes to visualize components.
• Applications: Studying interactions, structural/cellular changes, localization in cell/tissue samples

Fluorescence Microscopy (FM)

• Techniques: Combining antibodies and fluorescent markers
• Fluorochromes: Bind to and identify specific targets/antigens; Excitation-emission spectrum pairs for better clarity.
• Advantages: Powerful technique for intracellular process visualisation, and very similar in principle to other techniques.
• Disadvantages: Expensive equipment required.

Western Blotting

• Techniques & Methods
  • Separates proteins by size.
  • Transfers proteins to membrane.
  • Detects specific proteins with antibodies.

PCR (Polymerase Chain Reaction)

• A method for exponentially amplifying DNA segments.
• Denaturation: High temperature - separating double-stranded DNA into single strands
• Annealing: Lowering temperature—allowing primers to bind to the DNA
• Extension: Maintaining a higher temperature—allowing DNA polymerase to synthesize new strands.

qPCR (Quantitative PCR)

• Identifies how much of a specific gene is in a sample; used in Real-time PCR
• Quantification methods (Absolute and Relative)
• Absolute method: compares CT values of test samples vs. optimized curve
• Relative method: normalizes values to an internal control

Melt Curve Analysis

• Assesses product specificity; helps to identify whether the primer(s)/reaction is (are) working.
• Single peak = high specificity
• Multiple peaks = possible contamination, poor design, or other factors.

PFAFFL Method

• Does not require amplification of both genes to be 100% equal.
• Considers the efficiencies of specific gene amplifications.

Blood Collection/Erythrocytes

• Teleost fish: Lateral approach near lateral spine.
• Elasmobranchs: Caudal venous plexus or occipital plexus.
• Sea turtles: Jugular, occipital, or sub-carapacial veins.

Hemogram (Complete Blood Count, CBC)

• Measures cells like erythrocytes, leukocytes, and platelets and their morphology.
• Aids in diagnosis, treatment, and monitoring of health.

Cell Count Methods (Quantitative)

• Manual methods (Neubauer chamber): Less accurate, high variability.
• Automatic methods: Accurate and more reproducible methods, often electronic.