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Пишет bioRxiv Subject Collection: Neuroscience (![[info]](http://lj.rossia.org/img/syndicated.gif){kind=small} syn_bx_neuro)@ 2024-12-11 09:25:00 |
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Collablots: Quantification of collagen VI levels and its structural disorganisation in cell cultures from patients with collagen VI-related dystrophies
AimsThis 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.
MethodsWe developed a combined in-cell western (ICW) and on-cell western (OCW) assay, that 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.
ResultsCollablots 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.
ConclusionsThe collablot assay represents an optimal method for quantifying collagen VI expression and its organisation in culture and assessing the effect of therapies.
Key Points- Evaluating therapies for collagen VI-related dystrophies (COL6-RD) requires the quantification of collagen VI levels.
- Collablot assays are a novel method for quantifying collagen VI expression and its structural organisation in cell culture.
- Due to the significant role of phenotype heterogeneity in this complex disease, quantifying collagen alone might not be adequate for diagnosing COL6-RD, but the addition of a peptide to quantify collagen disorganisation could help in the characterisation of patient cultures.
AimsThis 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.
MethodsWe developed a combined in-cell western (ICW) and on-cell western (OCW) assay, that 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.
ResultsCollablots 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.
ConclusionsThe collablot assay represents an optimal method for quantifying collagen VI expression and its organisation in culture and assessing the effect of therapies.
Key Points- Evaluating therapies for collagen VI-related dystrophies (COL6-RD) requires the quantification of collagen VI levels.
- Collablot assays are a novel method for quantifying collagen VI expression and its structural organisation in cell culture.
- Due to the significant role of phenotype heterogeneity in this complex disease, quantifying collagen alone might not be adequate for diagnosing COL6-RD, but the addition of a peptide to quantify collagen disorganisation could help in the characterisation of patient cultures.
