Chapter 37. Biology Experiment
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b. Transmission Electron Microscope
d. Pharmacology(PK/PD)
g. Types of Fluorescent Substances Used in Biological Experiments
h. Abbreviations Related to Biology Experiments
1. Quantitative Experiment
⑴ Method 1. Centrifugation
① Cell Fractionation Method
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 : Iso-density separation
③ Sucrose Concentration Gradient Centrifugation
○ Principle : Direct movement. Lower partition has higher density
○ Advantages : Actual collection can be done
⑵ Method 2. Absorption Quantification
① Example 1. Colorimetric lactate quantification : Measurement of lactate content
⑶ 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
② 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
③ FRET (Förster/fluorescence resonance energy transfer)
○ Purpose : Proximity assessment of two proteins
○ Principle : Fluorescence resonance energy transfer
⑷ Method 4. Radiometric Quantification
① Hydrogen (3H) : Nucleic acid quantification
○ [3H]-dT (deoxycytidine) : Compound using radioactive isotope to label cellular DNA
○ Pulse-chase : Method to observe the difference after attaching radioactive label for a certain time and then removing it
○ Pulse-labeling : Method to continuously attach radioactive label for observation
○ [3H]-UDP : Targets the site where transcription occurs
② Carbon (16C) : Diagnosing cancer by tracking glucose
③ Fluorine (18F) : PET-CT scan using FDG
④ Phosphorus (32P) : DNA tracking
○ α-32P in ATP : Radioactive isotope attached to ATP’s α position
○ Tracing phosphate ester linkage (skeleton of DNA and RNA)
○ Verifying presence of nucleotides
○ γ-32P in ATP : Radioactive isotope attached to ATP’s γ position
○ Tracing phosphate group in signal transduction
○ Confirming polymerization reaction
⑤ Sulfur (35S) : Protein tracking
⑥ Technetium (99mTc) : Nuclear medicine test over 70%
⑦ Indium (111In) : Brain tumor imaging
⑧ Iodine (123I) : Thyroid disorder test
⑨ Iodine (124I) : PET imaging
⑩ Iodine (125I) : In vitro sample test, thyroid treatment
⑪ Iodine (131I) : Thyroid tumor treatment
⑫ Thallium (201Tl) : Heart disease test
⑸ 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
○ Uses color change when treated with alkaline copper to measure absorbance
○ 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
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
⑹ Application 2. Nucleic Acid Content Analysis Method
① Hoechst 33258 method
○ Measures fluorescence generated by interaction between hoechst 33258 and DNA at 458 nm
○ Can measure up to 10 ng/ml, uses intact ds DNA
② DAPI(diamidino-2-phenylindole) method
○ Measures fluorescence at 454 nm
○ Binds to AT region of DNA minor grooves
○ Also used as apoptosis marker
③ UV 260 nm absorbance
○ Data 1.
○ OD of ds DNA at 1.0 corresponds to 50 μg/ml
○ OD of ss DNA at 1.0 corresponds to 40 μg/ml
○ Data 2. Absorbance increase phenomenon
○ ds DNA : Set at 1.0 and represent absorbance of other nucleic acids as relative values
○ ss DNA : 1.37
○ Modified nucleotide : 1.5
○ Only purified DNA is used due to interaction with proteins
④ Ribo Green Assay
○ Quantifies concentration of RNA in solution
⑺ Application 3. Immune Analysis Method : Immune precipitation, Radioimmune analysis, Immunohistochemistry, 3D immunostaining, etc.
⑻ Application 4. Binding Assay : Method to measure affinity between target and targeting agent
② Surface Plasmon Resonance
③ Isothermal Titration Calorimetry
④ Gel Chromatography
⑥ X-ray Crystallography
⑦ Cryo-Electron Microscopy
2. Cell Experiment
⑴ Microscope
② Scanning Electron Microscope (SEM) : Surface structure observation
③ Transmission Electron Microscope (TEM) : Internal structure observation
④ Scanning Tunneling Microscope (STM) : Surface structure observation
⑤ Atomic Force Microscope (AFM)
⑥ Dark-Field Microscope
⑦ Fluorescence Microscope
① Type 1. Transwell-Based Model
○ Migration
○ Invasion
○ Transendothelial Migration
② Type 2. Spheroid-Based Model
○ Cell Suspension Culture
○ Non-Adherent Surface
○ Hanging Drop Technique
○ Microfluidic Device
③ Type 3. Hybrid Model
○ Embedded Ex Vivo Tumor Section
○ 3D Invasion Model
○ Avascular Microfluidic Model
④ 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
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 Single-Cell Analysis Method (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
① Cell Toxicity Experiment : XTT assay, WST assay, XTT assay, CCK-8 assay, etc.
② Oil Red O Staining : Evaluation of adipocyte differentiation
③ [Alizarin Red S Staining](https://jb243.github.io/pages/447#:~:text=%EB%82%98.-,ARS%20%EC%97%BC%EC%83%89(,-Alizarin%20red%20s)(alizarin red S staining) : Evaluation of osteoblast differentiation
④ DCF Staining (Dichlorofluorescein Assay) : ROS Evaluation
⑤ Nissl Staining (Nissl Body)
○ Nissl bodies and rough endoplasmic reticulum stained, giving a leopard-like pattern
○ Alkaline stains are used for Nissl staining
⑸ DNA Technology
③ PCR (Polymerase Chain Reaction)
⑤ Homogenization : Southern Blotting, Northern Blotting, Western Blotting, DNA chip (microarray), ISH, etc.
⑥ Gene Mutation : Knockout mouse, Cre-Lox, siRNA
⑦ Nuclear Transplantation : Nuclear transfer, transgenesis, Genetically modified foods
⑧ DNA-Protein Interaction Study : Genetic fingerprinting, EMSA, ChIP, etc.
⑨ Protein-Protein Interaction Study : Heteroduplex system, yeast two-hybrid, etc.
⑩ Gene Therapy : CRISPR/Cas9 gene editing technology, siRNA therapy, mRNA drug delivery system, etc.
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
3. Tissue Experiments
⑴ Tissue Observation
① H & E Staining
○ Overview
○ H refers to hematoxylin, a basic stain, and E refers to eosin, an acidic stain.
○ H&E staining is a standard method used in clinical pathology to identify patients’ diseases or treatment methods.
○ Step 1: Fixation
○ Generally done with 10% neutral buffered formalin to prevent tissue autolysis or microbial decay.
○ Step 2: Gross Sectioning
○ Cutting the tissue into appropriate sizes or shapes.
○ Step 3: Washing
○ Step 4: Tissue Processing
○ Step 4-1: Dehydration : Removing water from the tissue.
○ Step 4-2: Clearing : Replacing alcohol used in dehydration with xylene.
○ Step 4-3: Infiltration : Filling the tissue with paraffin.
○ Step 5: Embedding
○ Creating paraffin blocks for tissue sectioning.
○ An embedding center is used.
○ Step 6: Sectioning
○ Cutting the tissue into thicknesses suitable for microscopic observation.
○ A microtome is used.
○ Step 7: H&E Staining & Mounting
○ Staining the tissue sections with H&E and covering them with cover glass for microscopic observation.
○ Nuclei : stained purple by hematoxylin
○ Cytoplasm : stained pink by eosin
○ Automated devices like autostainers are commonly used for H&E staining.
Figure. 2. H&E Staining
○ Step 8: Histopathological Interpretation
② Other Tissue Staining Techniques
○ Immunohistochemistry (IHC) Staining
○ ALP Assay (Alkaline Phosphatase Assay)
○ ALP is an enzyme commonly found in the liver and bone.
○ Hydrolyzes phosphate groups under alkaline conditions (pH 10.5).
○ Measured by absorbance at 405 nm.
○ Masson’s Trichrome Staining
○ Red : Cytoplasm, keratin, muscle fibers, red blood cells
○ Black : Nuclei
○ Blue : Collagen, mucin, reticular fibers
○ PAS (Periodic Acid-Schiff) Staining
○ Special staining for observing purple glycogen components.
○ Can also observe other polysaccharides and mucosubstances (mucin, glycoprotein, etc.).
○ 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 for evaluating cell pathology and brain protein integrity.
○ Herovici staining : It labels collagen deposits.
⑵ 3D Imaging Acquisition
① Intravital Imaging
○ Experimental process
Figure. 3. Intravital Imaging Experimental Process
○ GSL-1-cy3 is used for intravital imaging of blood vessel walls.
○ US : Assessment of health status using the frequency difference between emitted and reflected ultrasound waves in a diagnostic device.
○ PET : 3D non-invasive imaging device utilizing positron emission from isotopes that collide and emit gamma rays upon encountering electrons.
○ MRI : Non-invasive imaging of magnetic relaxation in living organisms or samples using nuclear magnetic resonance.
○ CT : X-ray-based 3D imaging where the darker image corresponds to higher X-ray penetration.
○ SPECT : 3D non-invasive imaging using gamma-emitting isotopes for imaging, similar to X-ray CT.
○ TPEM
⑶ Tissue Toxicity Experiments
① Intravenous Reaction
○ Evaluation of local irritability after intravenous injection of test substances.
○ Used when applying irritability tests in animal experiments is not feasible or when the test substance is lipophilic.
② Hemolysis Assay
○ Experiment to assess red blood cell dissolution and hemoglobin release.
○ Steps: 1st - Blood addition to EDTA-containing vacuum blood collection tube, 2nd - 1-hour incubation followed by centrifugation, 3rd - Measurement of hemoglobin release, 4th - Hemolysis rate calculation.
③ Platelet Aggregation Assay
○ Type 1: Measurement of Platelet Count
○ Type 2: Platelet Aggregation
○ Type 3: Measurement of Blood Cell Adhesion : Lower adhesion indicates higher blood compatibility.
④ Immunological Tests
○ 1st - Peripheral Blood Mononuclear Cells (PBMCs) encounter foreign substances and trigger inflammatory response leading to cytokine production.
○ 2nd - Production levels assessed through reverse transcription-polymerase chain reaction or enzyme-immunoassay methods.
⑤ Plasma Protein Coagulation Assay
○ Test of plasma protein characteristics on material surfaces.
○ Plasma Proteins: Albumin, globulin, fibrinogen, immunoglobulin, etc.
○ Types of Plasma Protein Coagulation Assays
○ Partial Thromboplastin Time (PTT)
○ Prothrombin Time (PT)
○ Thrombin Time (TT)
○ Fibrinogen
○ Fibrinogen and Fibrin Degradation Products (FDP)
○ Specific Coagulation Factor Measurement Methods
○ FPA, D-dimer, F1+2, TAT
○ Lee-White Method
○ Imai-Nose Method
① ISS ( in situ sequencing)
② Spatial Transcriptomics
4. Animal Experiments
⑴ Overview : Corresponds to preclinical experiments
⑵ General Process of Animal Experiments
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
① Study of biochemical reactions related to drug and drug administration
② Divided into Pharmacodynamics (PD) and Pharmacokinetics (PK)
5. Clinical Trials : Involves humans. Typically performed sequentially: cell experiments → animal experiments → clinical trials.
⑴ Overview
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.
⑵ Phase 1 Clinical Trials : Exploratory clinical trials
① Involving 20 ~ 80 participants
② Type 1: If the purpose is safety assessment, healthy volunteers are used.
③ Type 2: Anti-cancer drugs : Tested on a small number of terminal cancer patients.
④ Objectives
○ Pharmacokinetics (ADME) theory
○ 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
⑶ 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
⑷ 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)
⑸ 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