WT and Hsf1 null primary colon fibroblasts were seeded at 2 × 105 cells/ml in 96-well plates. 24 h later (when the cells have formed a monolayer) the medium was replaced with either the same medium (as control) or with MC38 condition medium (prepared as described above) supplemented with collagen secretion medium57 (link) (l -ascorbic acid (cat. A8960, Sigma-Aldrich, Rehovot, Israel) 7.5 mg/ml; h-insulin (cat. 12585-0147.5, Gibco, NY, USA) 0.25 mg/ml; m-EGF (cat. PMG804, Gibco, NY, USA) 0.25 ng/ml. Half of the medium was changed every day, for 5 days, after which cell lysates were removed with acetic acid 0.5 M, and Sirius Red total collagen detection assay kit (cat. 9062, Chondrex, WA, USA) was used according to manufactures’ protocol to measure fibrillar collagen content.
Sirius red total collagen detection assay kit
Manufactured by Chondrex
Sourced in United States
The Sirius Red total collagen detection assay kit is a laboratory tool designed to quantify the total amount of collagen present in a sample. The kit utilizes the Sirius Red dye, which specifically binds to collagen, allowing for colorimetric detection and measurement of the collagen content.
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2 protocols using sirius red total collagen detection assay kit
1
Colon fibroblasts collagen secretion
2
Quantification of Tissue Components
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Generated tissues were digested for 16 h at 56 °C in proteinase K [1 mg/mL proteinase K (P2308, Sigma-Aldrich) in 50 mM Tris (A5456-3, Sigma-Aldrich) with 1 mM ethylenediaminetetraacetic acid (EDTA; 03680, Fluka), 1 mM iodoacetamide (I-1149, Sigma-Aldrich) and 10 mg/mL pepstatin A (P5318, Sigma-Aldrich)]. Glycosaminoglycan (GAG) content was determined spectrophotometrically using dimethyl methylene blue, with chondroitin sulphate as standard (Barbosa et al., 2003) . DNA content was measured using the CyQuant cell proliferation assay kit (Invitrogen), with calf thymus DNA as standard.
Collagen assays were performed using Sirius Red Total Collagen Detection Assay Kit (9062, Chondrex), Human type I Collagen ELISA detection Kit (6021, Chondrex) and Multi-Species Type II Collagen ELISA Detection Kit (6018, Chondrex). All three assays were performed on the same samples. Samples were solubilised and processed according to the manufacturer's instructions.
Collagen assays were performed using Sirius Red Total Collagen Detection Assay Kit (9062, Chondrex), Human type I Collagen ELISA detection Kit (6021, Chondrex) and Multi-Species Type II Collagen ELISA Detection Kit (6018, Chondrex). All three assays were performed on the same samples. Samples were solubilised and processed according to the manufacturer's instructions.
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