Neuropathology and applied neurobiology · 2025
Collablots: Quantification of Collagen VI Levels and Its Structural Disorganisation in Cell Cultures From Patients With Collagen VI-Related Dystrophies.
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Osegui-Barcenilla Nadia, Sendino Maria, Martín-González Sergio, González-Moro Itziar, Benito-Agustino Ainhoa, Torres-Conde Noemi, López-Martínez Andrea, Jiménez-Mallebrera Cecilia, López-Márquez Arístides, Arechavala-Gomeza Virginia
Аннотация
AIMS: This study aims to develop a quantitative method for assessing collagen VI expression in cell cultures, which is crucial for the diagnosis and treatment of collagen VI-related dystrophies.
METHODS: We developed a combined in-cell western (ICW) and on-cell western (OCW) assay, which we have called 'collablot', to quantify collagen VI and its organisation in the extracellular matrix of cell cultures from patients and healthy controls. To optimise it, we optimised cell density and the protocols to induce collagen expression in cultures, as well as the cell fixation and permeabilisation methods. This was completed with a thorough selection of collagen antibodies and a collagen-hybridising peptide (CHP). We then used collablots to compare cultures from patients and controls and evaluate therapeutic interventions in the cultures.
RESULTS: Collablots enabled the quantification of collagen VI expression in both control and patient cells, aligning with immunocytochemistry findings and detecting variations in collagen VI expression following treatment of the cultures. Additionally, CHP analysis revealed a marked increase in collagen network disruption in patients compared to the controls.
CONCLUSIONS: The collablot assay represents a suitable method for quantifying collagen VI expression and its organisation in culture and assessing the effect of therapies.
METHODS: We developed a combined in-cell western (ICW) and on-cell western (OCW) assay, which we have called 'collablot', to quantify collagen VI and its organisation in the extracellular matrix of cell cultures from patients and healthy controls. To optimise it, we optimised cell density and the protocols to induce collagen expression in cultures, as well as the cell fixation and permeabilisation methods. This was completed with a thorough selection of collagen antibodies and a collagen-hybridising peptide (CHP). We then used collablots to compare cultures from patients and controls and evaluate therapeutic interventions in the cultures.
RESULTS: Collablots enabled the quantification of collagen VI expression in both control and patient cells, aligning with immunocytochemistry findings and detecting variations in collagen VI expression following treatment of the cultures. Additionally, CHP analysis revealed a marked increase in collagen network disruption in patients compared to the controls.
CONCLUSIONS: The collablot assay represents a suitable method for quantifying collagen VI expression and its organisation in culture and assessing the effect of therapies.