Groups of mice were sacrificed at 12 weeks post-infection and lungs were removed and homogenized as describe above. Isolation and concentration of total collagen was performed using the homogenized suspension with acid neutralizing reagent (0.5M acetic acid, 0.1 mg/ml pepsin) (Sigma-Aldrich, Saint Louis, MO, USA) and collagen isolation & concentration reagent (Biocolor, Northern Ireland, U.K.) and centrifuged at 12,000 rpm, 4°C, 10 min. Then, the pellets were resuspended with Sircol Dye reagent (Biocolor, Northern Ireland, U.K.) and proceed with the Sircol Soluble Collagen Assay according to the manufacturer instructions.
Sircol dye reagent
Manufactured by Biocolor
Sourced in United Kingdom
The Sircol Dye Reagent is a laboratory product designed for the quantitative determination of soluble collagen. It is a colorimetric assay that measures the amount of collagen present in a sample by binding the Sircol dye to the collagen molecules. The Sircol Dye Reagent provides a simple and reliable method for the measurement of collagen in various biological samples.
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Lab products found in correlation
6 protocols using sircol dye reagent
1
Isolation and Quantification of Lung Collagen
2
Quantifying Soluble Collagen in Cell Cultures
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The total soluble collagen content of ECs and SCs in monocultures was evaluated using the Sircol™ Soluble Collagen Assay (Biocolor, Carrickfergus, United Kingdom) measuring mammalian type I-V collagen at the d 14 time point, as described previously [18 (link), 34 ]. Briefly, the samples were incubated in 0.5 M acetic acid (Merck, Darmstadt, Germany) with 0.1 mg/ml pepsin (Sigma-Aldrich) for 4 h at + 4 °C to extract the acid-soluble collagen from samples. Sircol Dye reagent (Biocolor) was added to liquid samples and incubated with gentle shaking for 30 min at RT. The samples were centrifuged for 10 min at 12 000 rpm, and collagen pellets were washed with ice-cold Acid-Salt Wash Reagent (Biocolor). The samples were centrifuged again, and the dyed collagen was further dissolved in 0.5 M sodium hydroxide solution (Biocolor), and the intensity of the red dye was measured with a Victor 1420 microplate reader (Wallac) at 540 nm.
3
Quantification of Soluble GAG and Collagen
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Soluble sulphated glycosaminoglycan (sGAG) standards, blanks and conditioned culture medium samples were mixed with the sGAG-binding Blyscan Dye Reagent® for 30 minutes, followed by separation of the GAG–dye complex by centrifugation and dissociation of the dye from the pellet (Biocolor Ltd., Carrickfergus, UK). Soluble Col-II was similarly mixed with the collagen-binding Sircol Dye Reagent® (Biocolor Ltd.) for 30 minutes, followed by centrifugation, pelleting, washes with salt wash reagent, centrifugation and dissociation of the collagen–dye complex using an alkali reagent vortexing wash. Absorbance was measured at 595 and 540 nm, respectively, using a plate reader (Chameleon).
4
Collagen Quantification in Estradiol-Treated Cells
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Cells (3.0 × 105 per well) were seeded in a 6-well plate and incubate in DMEM with 10% FBS. Cells were allowed to adhere to the plate overnight, and then the medium was changed to DMEM (2 ml per well) containing 17β-estradiol (10-7 < 10-9 M) with 2% FBS. After 10 days, 200 μl sample medium of each group was transfer to a 1.5 ml centrifuge tube and mixed with 1.0 ml Sircol Dye Reagent (Biocolor, Newtownabbey, Northern Ireland). The tubes were placed in a gentle mechanical shaker for 30 minutes and then centrifuged at 12,000 rpm for 10 minutes. The supernatant was discarded. The unbound dye was removed from the surface of the pellet and inner surface of the tube by rinsing with Acid-Salt Wash Reagent. The remaining pellets were mixed with 250 μl of alkali reagent and 200 μl of each sample was transferred to each well of a 96-well plate to measure the absorbance at 555 nm.
5
Quantification of Collagen and Elastin in Detrusor
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After myograph experiments, collagen and elastin content within the detrusor was determined. Mucosa was carefully removed, and detrusor was weighed. Then, 3 mg of detrusor was collected, and collagen was measured using Sircol™ Collagen Assay from Biocolor (Carrickfergus, UK) following the manufacturer's instructions. Briefly, tissue was minced using a fine scissor and digested with 0.1 mg/ml pepsin overnight at 4°C. This solution was centrifuged at 12,000 g for 10 min, and supernatant was carefully transferred to a clean tube and treated with an acid-neutralizing regent included in the assay kit. The collagen content was then determined using the Sircol Dye Reagent (Gray et al., 2008 (link); Toosi et al., 2008 (link)).
Similarly, elastin was measured using Fastin™ Elastin Assay (Biocolor) following the manufacturer's instructions. Elastin was extracted with 0.25 M oxalic acid at 100°C for 2 h and then precipitated using an Elastin Precipitating Reagent included in the assay kit. The elastin content was measured using the Elastin Dye Reagent (Gray et al., 2008 (link); Toosi et al., 2008 (link)).
Both collagen and elastin content were normalized to the wet tissue weight for each sample.
Similarly, elastin was measured using Fastin™ Elastin Assay (Biocolor) following the manufacturer's instructions. Elastin was extracted with 0.25 M oxalic acid at 100°C for 2 h and then precipitated using an Elastin Precipitating Reagent included in the assay kit. The elastin content was measured using the Elastin Dye Reagent (Gray et al., 2008 (link); Toosi et al., 2008 (link)).
Both collagen and elastin content were normalized to the wet tissue weight for each sample.
6
Quantifying Collagen Deposition in Cell Culture
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To visualise collagen deposition over time, Sirius red staining was performed at days 3, 5, 7 and 14. COBs were grown in 12 well plates at a density of 5 x 10 4 cells/well, with and without BGP and AA. After the desired culture period, cells were washed twice with PBS before fixing with 70% ethanol for 1 hour.
After fixation, cells were dried at 37°C and stained with Sircol Dye Reagent (Biocolor, County Antrim, UK) for 1 hour. After staining, cells were washed with dH 2 0 and allowed to air dry.
After fixation, cells were dried at 37°C and stained with Sircol Dye Reagent (Biocolor, County Antrim, UK) for 1 hour. After staining, cells were washed with dH 2 0 and allowed to air dry.
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