#trypsin

Animal Tissue Culture 2

Which cells do NOT require trypsin for passaging?

Adherent cell types such as fibroblasts, epithelial, endothelial, and myoblasts develop integrin-mediated focal adhesions and cadherin-based cell-cell contacts requiring proteolytic cleavage for detachment. Trypsin-EDTA treatment disrupts these connections enabling replating. Hematopoietic lineage cells including lymphocytes, monocytes, erythroblasts, mast cells, and numerous leukemia derivatives such as Jurkat, K562, HL-60 naturally grow in suspension, circulating in bloodstream and bone marrow without organized extracellular matrix attachment. They rely on cytokines like IL-2, IL-3, GM-CSF f

Ref: Freshney Ch.14 Suspension vs adherent cultures; Abbas Cellular and Molecular Immunology 10th Ed hematopoietic cells suspension growth.

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Animal Tissue Culture 2

EDTA enhances the activity of:

Ethylenediaminetetraacetic acid is polyaminocarboxylic acid with hexadentate chelation property exhibiting high affinity for divalent cations calcium and magnesium. Many cell adhesion molecules are calcium-dependent: classical cadherins require calcium to rigidify extracellular repeats for homophilic trans-interaction, and integrins need divalent ions for conformational activation and ligand binding to RGD motifs in extracellular matrix. By sequestering calcium, EDTA disrupts these homophilic bonds, loosens adherens junctions, desmosomes, and focal adhesions, enhancing penetration of trypsin t

Ref: Alberts MBoC 6th Ed Ch.19 Cadherin Ca2+ dependent adhesion; Freshney EDTA chelates Ca2+ enhances trypsin activity for cell detachment.

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Animal Tissue Culture 2

Enzymes commonly used for tissue disaggregation include:

Conversion of solid animal tissue into single cell suspension requires enzymatic disaggregation targeting extracellular matrix and intercellular junctions. Trypsin, serine protease from porcine pancreas, hydrolyzes peptide bonds on carboxyl side of basic residues lysine and arginine abundant in adhesion proteins fibronectin, laminin, and cadherin extracellular domains. Collagenase, derived from Clostridium histolyticum, specifically cleaves triple-helical native collagen rich in proline-hydroxyproline-glycine repeats found in connective tissue, basement membrane, and cartilage, requiring calci

Ref: Freshney Culture of Animal Cells Ch.11 Primary Culture Trypsin-Collagenase; Lodish Molecular Cell Biology 8th Ed ECM degradation enzymes.

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Animal Tissue Culture 1

Trypsin is mainly used in animal cell culture for:

Adherent mammalian cells deposit organized extracellular matrix and engage tissue culture surface through integrin mediated focal adhesions linking actin cytoskeleton to substrate, providing survival signaling through focal adhesion kinase FAK autophosphorylation tyrosine 397 recruiting Src, paxillin, vinculin, plus cadherin mediated cell-cell contacts dependent on calcium bridging extracellular domains EC1-EC2 homophilic binding. Subculture requires breaking these adhesions without irreversible damage to surface receptors. Trypsin serine protease belonging to PA clan S1 family purified from b

Ref: Freshney trypsinization Arg Lys cleavage integrin cadherin detachment; Alberts MBoC serine protease catalytic triad EDTA synergy.

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