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#cell lysis

2 public questions tagged with this topic.

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 same light dose. This suicide approach enriches for label-retaining slow-cycling stem cells and enables purification of G0 fraction for functional assays. MTT assay alone measures viability without selective elimination, caspase induction causes widespread death irrespective of proliferative status, and chromium-51 release assay monitors cytotoxic T lymphocyte mediated membrane lysis quantifying target cell killing but not specifically targeting S phase cells. Hence BrdU plus light provides unique tool for functional separation based on DNA replication status in heterogeneous cultures and developmental systems.

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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DNA & RNA isolation

Which method uses high frequency sound waves to shear cells during lysis?

Ultrasonication is a mechanical lysis method that employs high-frequency sound waves, typically above 20 kHz, to disrupt cells. Acoustic cavitation generates localized microbubbles in the suspension that implode violently, producing intense shear forces, shock waves, and transient high pressure that rupture rigid cell walls and lipid membranes. This efficiently releases intracellular nucleic acids into buffer. In contrast, freeze-thaw relies on intracellular ice crystal formation, bead milling uses physical collision with glass beads, and liquid homogenization uses pressure-driven shear through a narrow gap, none involving ultrasonic cavitation.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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