Fibrogenesis

fibrogenesis

Collagen formation assay
Collagen forms the ground substance of connective tissue. It is composed of three amino- acids and stains strongly with acid red dyes due to the affinity of the cationic groups of the proteins for the anionic reactive groups of the acid dyes. Activated HSC produces a large amount of collagen, Source: LRSU-AUKBC which is one of the important markers for HSC activation.
Collagen formation assay is performed using the collagen specific dye, Sirius red, as previously described (Lee et al 1998 and Marotta et al 1985). A solution of 5 mg/ml concentration Sirius red is prepared by dissolving 0.1g of Sirius red in 100ml of saturated picric acid solution. Cells are treated with drug for 8h (standardization for individual drug) and then probed with acidic solution of Sirius red.
cpllagen After 1h of incubation, cells are washed with PBS and then observed under microscope followed by capturing bright field images (refer figure on the left side, source LRSU-AUKBC). Colorimetric measurement of collagen bound Sirius red is performed by lysing the cells in sodium hydroxide (NaOH) 0.1M for 30min at room temperature and by measuring the supernatant at 530 nm using ELISA microplate reader.
Western blot analysis to measure collagen type I: Cells are treated with drug followed by measurement of secreted collagen type 1 in media and within the cells. Total protein concentration is normalized using Biuret Assay. Proteins are detected by using 1:1000 dilution of collagen type 1 antibody and 1:1000 dilutions of goat anti-rabbit IgG conjugated to horseradish peroxidase. The blots are developed by using substrate TMB/H2O2. Loading control used is β-actin as house keeping gene. (Majumder et al 2014).

REFERENCES

1. Marotta M, Martino G – Sensitive spectrophotometric method for the quantitative estimation of collagen. Anal Biochem.;150:86-90, 1985.
2. Lee, D.A., Assoku, E.,Doyle,V.,1998. A specific quantitative assay for collagen synthesis by cells seeded in collagen-based biomaterials using sirius red F3B precipitation. J.Mater.Sci.Mater.Med.9(1),47–51.
3. Majumder S, Gajalakshmi P, Chatterjee S. “Use of stem cells to block the activation of hepatic stellate cells in diseased liver” Edited by Somasundaram I in a book entitled “Stem cell therapy for organ failure” – Springer Verlag  Publications.2014 : 221-232.