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Chapter 37. Biology Experiment

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1. Quantitative Experiment

2. Cell Experiment

3. Tissue Experiment

4. Animal Experiment

5. Clinical Trial


a. Optical Microscope

b. Transmission Electron Microscope

c. Immune Analysis Method

d. Pharmacology(PK/PD)

e. Microbiology Experiment

f. Cell Culture Protocol

g. Types of Fluorescent Substances Used in Biological Experiments

h. Abbreviations Related to Biology Experiments



1. Quantitative Experiment

Method 1. Centrifugation

① Cell Fractionation Method


image

Figure 1. Cell Fractionation Method

A represents nucleus, B represents chloroplasts, C represents mitochondria


○ 1st Centrifugation (1,000g, 10 minutes): Nucleus precipitates

○ 2nd Centrifugation (3,000g, 10 minutes): Chloroplasts precipitate

○ 3rd Centrifugation (20,000g, 10 minutes): Mitochondria precipitate

○ 4th Centrifugation (150,000g, 180 minutes): Vesicles precipitate

○ Sedimentation Coefficient S

○ Svedberg unit

○ Sedimentation coefficient by sucrose gradient

○ Simple summation does not hold

② CsCl2 Density Gradient Centrifugation: particles come to rest at their characteristic density.

③ Sucrose Concentration Gradient Centrifugation

○ Principle: Direct movement. Lower partition has higher density

○ Advantages: Actual collection can be done

Method 2. Absorption Quantification

Example 1. Colorimetric lactate quantification: Measurement of lactate content

Method 3. Fluorescence Quantification

① Major Fluorescent Substances

○ Alexa series: Has NHS moiety for amine coupling

○ Di series: Lipophilic dye

○ FITC: Coupled with amine

○ GFP: Protein emitting green fluorescence upon UV exposure

○ SITS: Fluorescent substance specifically labeling amino group

○ Syto60: DNA dye

○ SYTOX: Measures dead cell amount using green fluorescent substance binding to chromatin

Other Fluorescent Substances Used in Biological Experiments

ELISA (enzyme linked immunosorbent assay)

○ Definition: Method used for detecting and quantifying specific antibodies and antigens

○ Direct ELISA: Constructs a structure like BSA - Target Molecule - 1st Antibody - 2nd Antibody to observe fluorescence

○ BSA: Improves resolution

○ 1st and 2nd antibodies should be from different animal species ( To enhance immune reaction sensitivity)

○ Indirect ELISA: Constructs a structure like BSA - 1st Antibody - Target Molecule - 2nd Antibody to observe fluorescence

○ BSA: Improves resolution

○ 1st and 2nd antibodies should be from different animal species ( To enhance immune reaction sensitivity)

○ Disadvantages

○ Not automated, labor-intensive

○ Expensive due to needing different antibodies for each target molecule

○ Dependent on fluorescence, so auto-fluorescence might occur

FRET (Förster/fluorescence resonance energy transfer)

○ Purpose: Proximity assessment of two proteins

○ Principle: Fluorescence resonance energy transfer

Method 4. Radiometric quantification

① Hydrogen (3H): nucleic acid quantification

○ [3H]-dT (deoxythymidine): a compound used to radioactively label cellular DNA.

○ pulse–chase: apply radioactive labeling for a defined period, then stop labeling to observe subsequent changes.

○ pulse labeling: apply labeling continuously and monitor.

○ [3H]-UDP: targets sites where transcription occurs.

② Carbon (16C): track glucose to diagnose cancer.

③ Fluorine (18F): FDG for PET–CT examinations.

④ Phosphorus (32P): DNA tracking

○ α-32P in ATP: radioisotope attached at ATP’s α position.

○ Track phosphate groups in phosphodiester bonds (the DNA/RNA backbone).

○ Check for the presence of nucleotides themselves.

○ γ-32P in ATP: radioisotope attached at ATP’s γ position.

○ Track phosphate groups in signal transduction.

○ Verify polymerization reactions.

⑤ Sulfur (35S): protein tracking.

⑥ Technetium (99mTc): used in >70% of nuclear-medicine examinations.

⑦ Indium (111In): brain-tumor imaging.

⑧ Iodine (123I): thyroid-disease testing.

⑨ Iodine (124I): PET imaging.

⑩ Iodine (125I): in-vitro specimen assays; thyroid therapy.

⑪ Iodine (131I): treatment of thyroid tumors.

⑫ Thallium (201Tl): cardiac testing.

Application 1. Protein Content Analysis Method

① Purpose: Measuring concentration of metabolites, receptor concentration, enzyme affinity

② Folin-Lowry method

○ Principle: Folin reagent reacts with aromatic amino acids like tyrosine, phenylalanine, tryptophan

○ Absorbance is measured by exploiting the blue color produced upon treatment with alkaline copper.

○ Range: 20 ~ 400 μg/ml

○ Disadvantage: Underestimates protein quantity if protein contains relatively fewer aromatic amino acids

③ Bradford method

○ Principle: Coomassie Blue G-250 binds to proteins under acidic conditions causing absorbance change

○ In dye-only condition, has 465 nm absorbance, while in protein + dye condition, has 595 nm absorbance

○ Advantages: Not dependent on aromatic amino acid content

○ Disadvantage: About 5 times more sensitive than Lowry method, so absorbance must be measured within 30 minutes

④ Biuret

○ Principle: Binding of Cu2+ with NH groups in proteins

○ Range: 1 ~ 20 mg/ml

○ Disadvantage: Rough method

BCA assay

⑥ UV 280 nm spectrometer

○ Principle: Uses 280 nm absorbance of phenyl groups in Phe, Trp, Tyr as representative

○ Disadvantage: Might have interference with DNA and about 10 times less sensitive than Lowry method

Application 2. Nucleic Acid Content Analysis Method

① Hoechst 33258 method

○ Measure fluorescence arising from the interaction of Hoechst 33258 with DNA at 458 nm.

○ Quantifiable down to 10 ng/mL, but requires intact double-stranded DNA (dsDNA).

DAPI(diamidino-2-phenylindole) method

○ Measure fluorescence at 454 nm.

○ Binds to AT-rich regions in DNA minor groove.

○ Also used as an apoptosis marker.

③ UV absorbance at 260 nm

Data 1.

○ OD 1.0 for dsDNA corresponds to 50 μg/mL.

○ OD 1.0 for ssDNA corresponds to 40 μg/mL.

Data 2. Hyperchromic effect

○ Set dsDNA = 1.0 and express other nucleic acids relatively

○ ssDNA: 1.37

○ Fragmented nucleotides: 1.5

○ Because proteins interfere, use purified DNA only.

④ RiboGreen assay

○ Quantifies the RNA concentration in solution.

Application 3. Immune Analysis Method: Immune precipitation, Radioimmune analysis, Immunohistochemistry, 3D immunostaining, etc.

Application 4. Binding Assay: Method to measure affinity between target and targeting agent

Yeast Two Hybrid Assay

② Surface Plasmon Resonance

③ Isothermal Titration Calorimetry

④ Gel Chromatography

NMR Spectroscopy

⑥ X-ray Crystallography

⑦ Cryo-Electron Microscopy

Radioligand Binding Assay



2. Cell Experiment

⑴ Microscope

Optical Microscope

② Scanning Electron Microscope (SEM): Surface structure observation

Transmission Electron Microscope (TEM): Internal structure observation

Scanning Tunneling Microscope (STM): Surface structure observation

⑤ Atomic Force Microscope (AFM)

⑥ Dark-Field Microscope

⑦ Fluorescence Microscope

Cell Culture

Type 1. Transwell-Based Model

○ Migration

○ Invasion

○ Transendothelial Migration

Type 2. Spheroid-Based Model

○ Cell Suspension Culture

○ Non-Adherent Surface

○ Hanging Drop Technique

○ Microfluidic Device

Type 3. Hybrid Model

○ Embedded Ex Vivo Tumor Section

○ 3D Invasion Model

○ Avascular Microfluidic Model

Type 4. Tumor-Microvessel Model

○ Predefined ECM Scaffold

○ Microvessel Self-Assembly

⑶ Cell Counting

① Hemocytometer

○ Originally used to count red and white blood cells

Cell Quantification Protocol using Hemocytometer

② Coulter Counter: Electronic Particle Counting

○ 1st. Cells are sucked through narrow pores, causing a change in current flow

○ 2nd. Change in current flow generates pulses

○ 3rd. Machine counts pulses to calculate cell number

○ Features: Can also do cell sizing, quantify ratio of living cells, aggregate cell quantification, not just cell counting

③ Stained Monolayer

○ Fixing cells directly on multi-well plate or Terasaki plate and staining for counting

○ Can be used when cell number is very low

④ Cell Weight

○ Used occasionally with inaccuracies when cell numbers are very high

Example 1. Murine Leukemia (e.g., L5178Y)

○ Diameter: 11-12 μm. Volume: 800 μm3

○ Cells/g × 106: 1250 (calculated), 1000 (measured)

Example 2. Hela

○ Diameter: 14-16 μm. Volume: 1200 μm3

○ Cells/g × 106: 800 (calculated), 250 (measured)

Example 3. Human Diploid Fibroblast

○ Diameter: 16-18 μm. Volume: 2500 μm3

○ Cells/g × 106: 400 (calculated), 180 (measured)

⑤ Flow Cytometry

○ Determines types and quantities of cells in cell suspension by discerning cells with specific antigens through antigen-antibody reactions

○ If the function of sorting desired cells is added, it’s called FACS (fluorescence-activated cell sorter)

⑷ Cell Staining

Cell Toxicity Experiment: XTT assay, WST assay, CCK-8 assay, etc.

Oil Red O Staining: Evaluation of adipocyte differentiation

Alizarin Red S Staining(alizarin red S staining): Evaluation of osteoblast differentiation

④ DCF Staining (Dichlorofluorescein Assay): ROS Evaluation

⑤ Nissl bodies (Nissl substance)

○ The ribosomes and rough (granular) endoplasmic reticulum are stained, producing a tigroid (leopard-like) pattern.

○ The dyes used for Nissl staining are basic (cationic) dyes.

⑥ Giemsa banding: A technique in which AT-rich heterochromatin stains dark and GC-rich euchromatin stains light; used for karyotype analysis.


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Figure 2. A male’s Giemsa banding photo


⑸ DNA Technology

DNA Recombination

Gene Library

PCR (Polymerase Chain Reaction)

DNA fingerprinting

Hybridization: Southern blotting, Northern blotting, Western blotting, DNA chip (microarray), ISH (in situ hybridization)

Gene deletion: knockout mouse, Cre-lox, siRNA

Nuclear transfer: nuclear transfer, transgenic organisms, genetically modified foods

DNA-protein interaction studies: genetic (DNA) fingerprinting, EMSA, ChIP

Protein-protein interaction studies: two-hybrid system, yeast two-hybrid

Gene therapy: CRISPR/Cas9 gene-editing technology, siRNA therapeutics, mRNA drug-delivery systems

in vitro Sequencing

① Sequencing Methods

○ in vitro Cloning

○ Dideoxy Chain Termination Method

○ Dye-Dideoxy Chain Termination Method

○ Pyrosequencing

○ Illumina Solid-Phase Amplification

② Sequencing Applications

○ WGS (Whole Genome Sequencing)

○ WES (Whole Exome Sequencing)

○ ChIP-seq

○ scRNA-seq (Single Cell RNA Sequencing)

○ Bisulfite Sequencing

○ Hi-C Sequencing

○ Long Read Sequencing

○ Non-invasive Sequencing

Microbiology Experiments



3. Tissue Experiment

⑴ Tissue Observation

H&E staining

○ Overview

○ “H” stands for hematoxylin, a basic dye; “E” stands for eosin, an acidic dye.

○ H&E staining is the standard method in clinical pathology for determining a patient’s disease and guiding treatment.

Step 1: Fixation

○ Typically performed with 10% neutral buffered formalin to prevent tissue autolysis and microbial putrefaction.

Step 2: Gross section (grossing)

○ Trimming the tissue to an appropriate size and shape.

Step 3: Washing

Step 4: Tissue processing

4-1. Dehydration: removing water from the tissue.

4-2. Clearing: replacing the alcohol used for dehydration with xylene.

4-3. Infiltration: permeating the tissue with paraffin.

Step 5: Embedding

○ Forming a paraffin block to enable thin sectioning; an embedding center is used.

Step 6: Sectioning

○ Cutting to a thickness suitable for microscopic observation; a microtome is used.

Step 7: H&E staining and mounting

○ Staining the paraffin sections with H&E and covering with a cover glass for microscopy.

○ Nuclei: stained purple by hematoxylin.

○ Cytoplasm: stained red by eosin.

○ Recently, automated devices such as autostainers are widely used for H&E.


image

Figure 3. H&E Staining


Step 8: Histopathological Interpretation

② Other Tissue Staining Techniques

Immunohistochemistry (IHC) Staining

○ ALP assay (alkaline phosphatase assay)

○ ALP is an enzyme found primarily in the liver and bone.

○ In an alkaline environment around pH 10.5, it hydrolyzes hydroxyapatite.

○ Measured by absorbance at 405 nm.

○ MT staining (Masson’s Trichrome staining)

○ Red: cytoplasm, keratin, muscle fibers, erythrocytes

○ Black: nuclei

○ Blue: collagen, mucin, collagen fibers

○ PAS (Periodic acid–Schiff) staining

○ A special stain used to visualize purple glycogen.

○ Can also detect other polysaccharides and mucosubstances (e.g., mucin, glycoproteins).

○ Jones’ Silver Stain: Staining for basement membrane

○ Sirius Red Staining: Special staining for observing red collagen components

○ Alcian Blue Staining: Staining for mucin

○ pH Map

○ DHE (Dihydroethidium) Staining: Detection of superoxide

○ Picrosirius Red Staining: ECM staining

○ Luxol fast blue: Used in cytopathology and to assess the integrity of cerebral white matter.

○ Herovici staining: Stains collagen deposits.

○ PHH3 staining (phospho-histone H3).

⑵ 3D Imaging Acquisition

① Intravital Imaging

○ Experimental process


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Figure 4. Intravital Imaging Experimental Process


○ GSL-1-cy3 is used for intravital imaging of blood vessel walls.

3D Immunostaining

Other 3D Imaging Devices

US: Assesses health status by using the frequency (Doppler) shift between ultrasound emitted by the probe and that reflected from blood.

PET: A three-dimensional, noninvasive imaging modality that detects pairs of γ rays produced when a positron-emitting radionuclide meets an electron and undergoes annihilation.

MRI: Uses nuclear magnetic resonance to noninvasively evaluate the magnetic relaxation properties of tissues or samples.

CT: X-ray–based tomography in which structures that transmit X-rays more readily appear darker; multiple projections around the subject are used to reconstruct a 3D image.

SPECT: Uses γ-emitting radionuclides for tomographic imaging, analogous in acquisition geometry to X-ray CT.

○ TPEM (two-photon excitation microscopy).

⑶ Tissue Toxicity Tests

① Intradermal Reaction

○ Evaluate local irritancy that appears after intradermal injection of the test article.

○ Used when animal-based irritancy tests are not applicable or when the test article is hydrophobic.

② Hemolysis Test

○ Assesses the degree of erythrocyte lysis and the release of hemoglobin.

○ 1st. Add blood to an EDTA-containing evacuated collection tube and incubate.

○ 2nd. After 1 hour of incubation, centrifuge.

○ 3rd. Collect the supernatant and measure the extent of hemoglobin release.

○ 4th. Hemolysis rate (%) = (Absorbance of test solution − Absorbance of blank) ÷ (Absorbance of positive control − Absorbance of blank) × 100.

③ Platelet Aggregation Test

Type 1: Platelet count.

Type 2: Platelet aggregation.

Type 3: Blood cell adhesion measurement: fewer adherent cells indicate higher blood compatibility.

④ Immunological Assays

○ 1st. Peripheral blood mononuclear cells (PBMCs) encounter foreign material, trigger an inflammatory response, and produce various cytokines.

○ 2nd. Quantify production by reverse-transcription PCR or enzyme-linked immunosorbent assay (ELISA).

⑤ Plasma Protein Coagulation Tests

○ Evaluate the behavior of plasma proteins on material surfaces.

○ Plasma proteins include albumin, globulins, fibrinogen, immunoglobulins, etc.

○ Types of plasma protein coagulation tests:

○ Partial thromboplastin time (PTT)

○ Prothrombin time (PT)

○ Thrombin time (TT)

○ Fibrinogen

○ Fibrinogen and fibrin degradation products (FDP)

○ Specific coagulation factor assays

○ FPA, D-dimer, F1+2, TAT

○ Lee–White method

○ Imai–Nose method

Hematology Tests

in situ Sequencing

① ISS ( in situ sequencing)

② Spatial Transcriptomics



4. Animal Experiments

⑴ Overview: Corresponds to preclinical experiments

⑵ General Process of Animal Experiments

Observation of Experimental Animals

Calibration, Administration, Blood Collection

Anesthesia Methods, Euthanasia, Autopsy

Toxicity Testing

Tumor Models

⑥ Regulations on Animal Experiments: Animal testing is generally prohibited for cosmetics.

⑶ Resources

① The Jackson Laboratory (ref1, ref2, ref3): Provides useful resources related to animal experiments

Pharmacology

① Study of biochemical reactions of body related to drug and drug administration

② Divided into Pharmacodynamics (PD) and Pharmacokinetics (PK)



**5. Clinical Trial

⑴ Overview: Involves humans. Typically performed sequentially: cell experiments → animal experiments → clinical trials.

① Drug Development Phases

○ Drug Discovery: 3 ~ 5 years

○ Preclinical: 1 ~ 2 years

○ Clinical Trials: 6 ~ 7 years

○ FDA Approval: 1 ~ 2 years

② Approximately 50 ~ 60 new drugs are approved by the FDA annually.

③ Increasing costs and a failure rate of around 90%.

④ Most failures are due to lack of therapeutic effect.

○ Efforts are made to prevent this by focusing on efficacy and dose finding even in Phase 1.

○ Phase 0 studies are introduced to administer drugs to humans more quickly.

○ PET imaging can also be used as a strategy.

Phase 1 Clinical Trials: Exploratory clinical trials

① Involving 20 ~ 80 participants

Type 1: If the purpose is safety assessment, healthy volunteers are used.

Type 2: Anti-cancer drugs: Tested on a small number of terminal cancer patients.

④ Objectives

○ Pharmacokinetics: Theory on ADME

○ Interactions

○ Safety (dose dependent)

○ Maximum Tolerated Dose (MTD), tolerable dose range, dose-response studies

○ PK / PD studies

⑤ Methods

○ Dose-response curve: NOAEL, NOEL, MED (Min Effective Dose), MABEL

○ Single dose rising, multiple dose rising

○ Drug-drug interaction

Phase 2 Clinical Trials: More participants are involved, exploratory clinical trials

① Involving 100 ~ 200 participants

② Early Phase 2 Clinical Trials (IIa): Assessing efficacy

③ Late Phase 2 Clinical Trials (IIb): Dose finding

Phase 3 Clinical Trials: Final stage of clinical trials for obtaining market approval. Confirmatory clinical trials

① Conducted on a large scale for statistical confirmation

② Objectives: Confirming safety and therapeutic effectiveness

③ Generally takes around 5 ~ 6 years until market approval (long-term)

Phase 4 Clinical Trials: Long-term assessment of new drug’s efficacy and safety after market use

① Post-marketing surveillance



Input: 2019.11.30 10:43

Last Update: 2022.11.07 22:51

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