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: 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
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
○ Ribogreen assay: Used when accurately quantifying the amount of RNAs
○ 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 (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.
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
○ 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
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
○ 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).
② 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.
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 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, CCK-8 assay, etc.
② Oil Red O Staining: Evaluation of adipocyte differentiation
③ 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.
Figure 2. A male’s Giemsa banding photo
⑸ DNA Technology
③ PCR (Polymerase Chain Reaction)
⑤ Hybridization: Southern blotting, Northern blotting, Western blotting, DNA chip (microarray), ISH (in situ hybridization)
⑥ Gene deletion: knockout mouse, Cre-lox, siRNA
⑦ Nuclear transfer: nuclear transfer, transgenic organisms, genetically modified foods
⑧ DNA-protein interaction studies: genetic (DNA) fingerprinting, EMSA, ChIP
⑨ Protein-protein interaction studies: two-hybrid system, yeast two-hybrid
⑩ Gene therapy: CRISPR/Cas9 gene-editing technology, siRNA therapeutics, mRNA drug-delivery systems
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 Experiment
⑴ Tissue Observation
① 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.
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
Figure 4. Intravital Imaging Experimental Process
○ GSL-1-cy3 is used for intravital imaging of blood vessel walls.
○ 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
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 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.
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: 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
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