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What is the purpose of denaturing proteins before running them on an SDS-PAGE gel?

To facilitate binding to antibodies

To increase their molecular weight

To allow them to run on the gel (correct)

To enhance their natural three-dimensional structure

What is the role of the stacking gel in SDS-PAGE?

To prevent proteins from escaping the gel

To act as a binding site for antibodies

To provide a higher concentration of acrylamide for low molecular weight proteins

To separate higher molecular weight proteins with a lower concentration of acrylamide (correct)

Why is it necessary to quantify proteins before loading them onto an SDS-PAGE gel?

To ensure the same amount of protein is loaded in each well (correct)

To increase the yield of proteins for analysis

To enhance the speed of electrophoresis

To decrease the risk of contamination

What method is used to transfer proteins from an SDS-PAGE gel to a membrane?

Semi-wet electrophoresis Signup and view all the answers

Which chemical agents are used for denaturing proteins in the provided protocol?

SDS and beta-mercaptoethanol Signup and view all the answers

What is the main difference between the running gel and stacking gel in SDS-PAGE?

The running gel has a higher concentration of acrylamide for separating lower molecular weight proteins Signup and view all the answers

What happens to proteins on the membrane after electro-transfer?

All proteins remain, including those not of interest Signup and view all the answers

Which of the following factors influences the migration of proteins during SDS-PAGE?

The molecular weight of the proteins Signup and view all the answers

What is the recommended maximum current for electrophoresis?

300mA Signup and view all the answers

For optimal results in electrophoresis, what condition must the electrophoresis tank meet?

It must be level. Signup and view all the answers

What is the purpose of placing slides in a PBS bath after electrophoresis?

To prevent crystal formation. Signup and view all the answers

Which of the following dyes is NOT used for staining slides after electrophoresis?

Methanol Signup and view all the answers

What does Class 0 represent in visual scoring for DNA damage?

Less than 5% Signup and view all the answers

What is the primary advantage of using computer-assisted image analysis over visual scoring?

It is more objective. Signup and view all the answers

In the Total Comet Score (TCS) calculation, what is the maximum possible score?

400 Signup and view all the answers

Which method allows for manual selection of comets during analysis?

Semi-automatic method Signup and view all the answers

What is a potential cause of non-specific bands in experimental results?

High concentration of antibodies Signup and view all the answers

What does a larger forward scatter cone indicate about a cell?

The cell is larger in size Signup and view all the answers

Which parameter can flow cytometry analyze in a single cell?

Up to 50 parameters Signup and view all the answers

Which of the following is NOT a factor that can affect background staining in experiments?

Proper blocking agent concentration Signup and view all the answers

What does side scatter in flow cytometry indicate?

Internal complexity and granularity Signup and view all the answers

Which part of flow cytometry data visualization typically follows a bimodal distribution?

Histogram of cells versus intensity pulse Signup and view all the answers

How is voltage pulse in flow cytometry quantified?

In terms of height, width, and voltage Signup and view all the answers

What common issue might arise from different protein levels being loaded into each assay?

The control protein may be invalid Signup and view all the answers

Study Notes

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.

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