Optimization of Sirius Red-Based Microplate Assay to Investigate Collagen Production In Vitro
Abstract
:1. Introduction
2. Results
2.1. Detection of Cell-Associated Collagens by Sirius Red Staining
2.2. Detection of Collagens in Cell Culture Medium by Sirius Red Staining
2.3. Optimization of In Vitro Experimental Setup to Detect Collagen Production of Fibroblasts
2.4. In Vitro Sirius Red Assay to Investigate the Efficacy of Antifibrotic Drugs
3. Discussion
4. Materials and Methods
4.1. Cell Lines and Treatments
4.2. Sirius Red Collagen Detection Assay for Cells
4.3. Sirius Red Collagen Detection Assay for Cell Culture Medium
4.4. Immunofluorescence Staining
4.5. Microscopy
4.6. Statistical Analysis
4.7. Step-by-Step Protocol of In Vitro Sirius Red Assay
4.7.1. Preparing Reagents
- Kahle’s fixative solution: 26% ethanol, 3.7% formaldehyde, and 2% glacial acetic acid diluted in distilled water
- Sirius Red solution for cell staining: 0.1% Direct Red 80 dissolved in 1% acetic acid containing distilled water
- Sirius Red solution for cell medium staining: 0.1% Direct Red 80 dissolved in 3% acetic acid containing distilled water
- HCl solution: 0.1 M HCl in distilled water
- NaOH solution: 0.1 M NaOH in distilled water
4.7.2. Cell Culture and Treatment
- seed cells into 96-well plates to reach near-full confluence and culture them for 24 h in a culture medium containing 10% FBS
- change to medium containing 1% FBS for 24 h to reach full confluence
- change to medium containing 0% FBS, 200 μM ascorbate, and treat cells with the examined agents or factors for 48 h
4.7.3. Sirius Red Staining of Cells
- transfer the cell culture medium into a V-bottom 96-well microplate
- carefully wash cells with 200 μL/well PBS
- fix cells with 50 μL/well Kahle’s solution at room temperature for 15 min
- wash cells with 200 μL/well PBS
- stain cells with 50 μL/well Sirius Red solution at room temperature for 1 h
- wash cells with 400 μL/well HCl solution
- elute collagen-bound dye with 100 μL/well NaOH solution
4.7.4. Sirius Red Staining of Cell Culture Medium
- add 50 μL/well Sirius Red solution to the previously transferred 100 μL/well medium, mix thoroughly, and incubate at room temperature for 30 min
- centrifuge the plates at 3000× g for 6 min to precipitate the collagen-dye complex from the solution
- remove the remaining fluid carefully without touching the precipitates
- add 150 μL/well HCl solution, then centrifuge the plates at 3000× g for 6 min
- remove the remaining fluid carefully without touching the precipitate
- elute collagen-bound dye with 100 μL/well NaOH solution
- transfer the eluted solution into transparent flat-bottom 96-well plates for detection
4.7.5. Detection
- measure the absorbance of the samples in a microplate reader at 540 nm wavelength
- either NaOH solution can be used as blank, or samples can be normalized on the background absorbance at 690 nm wavelength
- this method is applicable for the semi-quantitative comparison of collagen content between samples, but not for the exact quantification
- to quantify collagen content, collagen serial dilution in DMEM can be prepared and stained by the Cell Culture Medium protocol to create the standard curve
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Szász, C.; Pap, D.; Szebeni, B.; Bokrossy, P.; Őrfi, L.; Szabó, A.J.; Vannay, Á.; Veres-Székely, A. Optimization of Sirius Red-Based Microplate Assay to Investigate Collagen Production In Vitro. Int. J. Mol. Sci. 2023, 24, 17435. https://doi.org/10.3390/ijms242417435
Szász C, Pap D, Szebeni B, Bokrossy P, Őrfi L, Szabó AJ, Vannay Á, Veres-Székely A. Optimization of Sirius Red-Based Microplate Assay to Investigate Collagen Production In Vitro. International Journal of Molecular Sciences. 2023; 24(24):17435. https://doi.org/10.3390/ijms242417435
Chicago/Turabian StyleSzász, Csenge, Domonkos Pap, Beáta Szebeni, Péter Bokrossy, László Őrfi, Attila J. Szabó, Ádám Vannay, and Apor Veres-Székely. 2023. "Optimization of Sirius Red-Based Microplate Assay to Investigate Collagen Production In Vitro" International Journal of Molecular Sciences 24, no. 24: 17435. https://doi.org/10.3390/ijms242417435