Animal Tissue Culture 3

Animal Tissue Culture 3

EdU followed by doxorubicin treatment selectively targets:

Sequential application of EdU labeling followed by doxorubicin treatment represents strategy to track and preferentially eliminate newly dividing cells. EdU pulse marks cells traversing S phase with alkyne-modified nucleotide that persists in DNA of daughter cells, identifiable via click chemistry fluorescent azide. When doxorubicin subsequently administered, antineoplastic action via topoisomerase II trapping and generation of reactive oxygen species is most pronounced in actively cycling population where chromatin decondensed, replication forks active, and topoisomerase IIalpha expression hi

Ref: Thorn et al Doxorubicin S-phase targeting; Salic & Mitchison EdU newly dividing cells detection for chemosensitivity studies.

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Animal Tissue Culture 3

Selective lysis of dividing cells can be achieved by:

Selective lysis of dividing cells while sparing quiescent population can be accomplished using bromodeoxyuridine mediated photosensitization strategy. Cells pulsed with BrdU during S phase incorporate halogenated analog into newly synthesized DNA. Subsequent exposure to bright visible light or near-UV in presence of DNA binding dyes such as Hoechst 33258 sensitizes BrdU-substituted DNA to photolysis where carbon-bromine bond breaks generating uracilyl radicals causing strand breaks overwhelming repair and triggering apoptosis. Non-dividing cells lacking BrdU incorporation remain resistant to s

Ref: Poot et al J Histochem Cytochem 1991 BrdU photolysis selective lysis dividing cells; Thermo Fisher BrdU labeling followed by light selective killing.

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Animal Tissue Culture 3

The purple color in MTT assay is due to formation of:

Purple color appearing in wells after MTT incubation arises from formation of formazan product 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan. Enzymatic reduction transfers electrons from succinate via succinate dehydrogenase, NADH and NADPH to tetrazolium ring breaking positively charged quaternary nitrogen and producing neutral formazan with extensive conjugated double bond system delocalizing electrons, absorbing green-yellow wavelengths around 570 nm and appearing purple. Crystals observable microscopically as dark needle-like precipitates clustered around nucleus and in cytoplasm befor

Ref: Berridge et al Arch Biochem Biophys 2005 MTT formazan formation mechanism; Abcam MTT assay protocol formazan purple product succinate dehydrogenase.

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Animal Tissue Culture 3

MTT assay measures:

MTT assay developed by Tim Mosmann in 1983 measures cell viability and proliferation based on reduction capacity of living cells. Principle uses yellow tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide which enters cells via endocytosis and is reduced by mitochondrial succinate dehydrogenase of complex II and cytosolic NAD(P)H-dependent oxidoreductases cleaving tetrazolium ring with electrons from NADH and NADPH producing insoluble purple formazan crystals accumulating intracellularly in mitochondria and lipid droplets. Metabolically incompetent dead cells fail to r

Ref: Mosmann T J Immunol Methods 1983 MTT viability assay; ATCC MTT Cell Proliferation Assay mitochondrial dehydrogenase reduction protocol.

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Animal Tissue Culture 3

Doxorubicin induces cell death mainly by:

Doxorubicin, anthracycline antibiotic isolated from Streptomyces peucetius subsp. caesius, induces cytotoxicity via dual mechanisms of DNA damage and oxidative stress. Planar tetracyclic chromophore intercalates between DNA base pairs preferentially at CpG sequences increasing helical length and inhibiting topoisomerase IIalpha by trapping covalent enzyme-DNA cleavage complex preventing resealing of transient double-strand breaks introduced during replication, leading to activation of ATM kinase, phosphorylation of histone H2AX at serine139 forming gamma-H2AX foci, p53 accumulation, and mitoch

Ref: Thorn et al PharmGKB Doxorubicin topoisomerase II ROS; Gewirtz DA Biochem Pharmacol 1999 Doxorubicin DNA damage ROS mechanisms.

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Animal Tissue Culture 3

An advantage of EdU assay is:

Predominant advantage of EdU assay over BrdU is preservation of cellular architecture because it avoids harsh DNA denaturation. BrdU detection requires unwinding double helix using strong acid, heat, or enzymatic digestion to expose concealed bromo-deoxyuridine epitope for anti-BrdU antibody of large size 150 kDa that cannot access double-stranded DNA. These treatments hydrolyze fluorescent proteins such as GFP, destroy conformational epitopes of surface antigens CD markers, fragment histology, alter light scatter properties in flow cytometry, and distort cell cycle distributions. EdU click de

Ref: Buck et al Biotechniques 2008 EdU advantage no denaturation preserves antigens; Thermo Fisher Click-iT EdU vs BrdU no DNA denaturation needed.

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Animal Tissue Culture 3

EdU assay differs from BrdU assay because it:

EdU, 5-ethynyl-2'-deoxyuridine, retains Watson-Crick pairing but bears terminal alkyne at 5 position instead of bromine. Detection utilizes bioorthogonal click chemistry specifically copper(I)-catalyzed azide-alkyne cycloaddition first described by Sharpless. In this reaction, alkyne on EdU incorporated into DNA reacts with fluorescent azide forming stable 1,2,3-triazole covalent linkage, catalyzed by CuSO4 reduced to Cu(I) by sodium ascorbate, proceeding rapidly in aqueous buffer at room temperature with exquisite specificity. Because fluorescent azide small molecule penetrates duplex DNA, ha

Ref: Salic A & Mitchison TJ PNAS 2008 EdU click chemistry; Kolb et al Click chemistry concept Nobel Prize 2022 CuAAC azide-alkyne.

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Animal Tissue Culture 3

BrdU-labelled cells can be selectively killed by exposure to:

DNA containing BrdU substituted in place of thymidine becomes photosensitive because carbon-bromine bond cleaves homolytically upon absorption of near-UV light around 313 nm or visible light when sensitized by intercalating dyes Hoechst 33258 or acridine orange. Resulting uracilyl radical abstracts hydrogen from deoxyribose causing strand break at apurinic site, creating alkali-labile lesions and single- and double-strand breaks that overwhelm base excision repair leading to cell death if incorporation extensive. This property underlies selective elimination technique: pulse culture with BrdU

Ref: Davidson RL et al PNAS 1974 BrdU photosensitivity light killing; Thermo Fisher BrdU labeling followed by light UV selective cell death.

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Animal Tissue Culture 3

BrdU is a thymidine analog used to detect:

BrdU, 5-bromo-2'-deoxyuridine, is synthetic halogenated analog of thymidine where methyl group at 5 position replaced by bromine atom similar in size allowing Watson-Crick pairing with adenine during DNA synthesis. During S phase, DNA polymerases incorporate BrdU triphosphate from nucleotide pool into nascent strand in place of thymidine without halting polymerase at low micromolar concentrations. Incorporated BrdU detectable by specific anti-BrdU monoclonal antibodies after DNA denaturation exposes epitope, or by mass spectrometry. Assay quantifies fraction of cells actively replicating genom

Ref: Gratzner HG Science 1982 BrdU thymidine analog DNA replication detection; Alberts MBoC Ch.17 DNA synthesis S phase BrdU incorporation.

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Animal Tissue Culture 3

Generation time is defined as:

Generation time, synonymous with doubling time, quantifies interval needed for population to double during exponential phase, reflecting duration of one complete mitotic cycle comprising G1 phase where cells assess nutrient sufficiency and growth factor signaling via Ras-MAPK and PI3K-Akt inducing cyclin D, S phase where DNA polymerase alpha-primase and delta synthesize new genome with fidelity checks, G2 phase where mitotic proteins such as cyclin B and Cdk1 accumulate, and M phase involving spindle formation, chromosome segregation, and cytokinesis. Mathematically derived from growth curves

Ref: Freshney Ch.13 Generation time one division; Lodish MBoC Ch.13 Cell cycle timing G1 S G2 M doubling time calculation.

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Animal Tissue Culture 3

The senescence phase in cell culture corresponds to:

Senescence phase in cell culture corresponds to death or decline phase of growth curve where proliferative capacity permanently lost and net cell number decreases. Replicative senescence triggered by telomere attrition beyond critical threshold leads to uncapped chromosome ends recognized as DNA double-strand breaks activating ATM and ATR kinases that stabilize p53 and induce cyclin-dependent kinase inhibitors p21CIP1 and p16INK4a causing irreversible G1 arrest. Morphology changes to enlarged flattened cells with increased granularity, vacuolation, positive senescence-associated beta-galactosi

Ref: Hayflick L Exp Cell Res 1961 senescence death phase; Campisi J Cell 2005 Senescence SASP and p16 p53 pathways.

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Animal Tissue Culture 3

Cells should ideally be passaged during:

Growth curve of cultured cells comprises lag adaptation, exponential log proliferation, plateau where division balances loss, and death where loss predominates. Ideal timing for passaging aligns with transition from late log to early plateau when cultures achieve sufficient density providing high yield of cells for splitting but before deleterious effects of overconfluence arise. At this point confluence typically 70-80% for adherent fibroblasts and epithelial lines, or suspension density half of maximal sustainable concentration, providing many cells yet maintaining health with active DNA syn

Ref: Freshney Ch.14 Passaging at plateau late log; Alberts MBoC Ch.17 Growth regulation subculture timing before overconfluence stress.

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