The wet weights of the OA cartilage explants were determined (n = 6 per group). After grinding, explants were digested overnight in papainase (125 μg ml−1) at 60 °C. The GAG content was measured using a DMMB assay. Lysates (20 μl) were mixed with 200 μl of DMMB working solution for 30 min at room temperature. The absorbance was then measured at 525 nm. Chondroitin sulfate (Sigma) was used as a standard. The DNA content was determined using the Hoechst 33258 assay (Beyotime Biotechnology, Beijing, China). Briefly, 20 μl of lysate was mixed with 200 μl of Hoechst 33258 working solution and incubated at 37 °C for 1 h. Absorbance was determined at 360 nm for excitation and at 460 nm for emission. Calf thymus DNA (Sigma) was used as the standard. The GAG or DNA content was shown as micrograms of GAG or DNA per milligram of wet weight.
Hoechst 33258 assay
Manufactured by Beyotime
Sourced in China
Hoechst 33258 is a fluorescent dye that binds to adenine-thymine (A-T) rich regions of DNA. It is commonly used in biochemical assays to quantify DNA content.
Automatically generated - may contain errors
4 protocols using hoechst 33258 assay
1
Quantifying Cartilage Glycosaminoglycans and DNA
2
Characterization of Decellularized Mouse Skin
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From wild-type C57/B16 mice (Huafukang Co., Beijing) aged 5 days old, dermal homogenates were prepared by homogenizing freshly collected hairless mouse PD with isotonic phosphate buffer (pH 7.4) for 20 min in an ice bath to obtain 25% (w/v) tissue suspension. The supernatant was obtained after centrifugation at 4°C for 20 min at 10,000g. The DNA content was determined using Hoechst 33258 assay (Beyotime, Beijing). The fluorescence intensity was measured to assess the amount of remaining DNA within the decellularized ECMs and the native tissue using a fluorescence spectrophotometer (Thermo Scientific, Evolution 260 Bio, USA). The GAGs content was estimated via 1,9-dimethylmethylene blue solution staining. The absorbance was measured with microplate reader at wavelength of 492 nm. The standard curve was made using chondroitin sulfate A. The total COL (Collagen) content was determined via hydroxyproline assay. The absorbance of the samples was measured at 550 nm and quantified by referring to a standard curve made with hydroxyproline.
3
Hoechst 33258 Assay for Apoptosis
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Hoechst 33258 Assay was purchased from Beyotime Institute of Biotechnology (Shanghai, China). Hoechst 33258 is a blue fluorescent dye which is used for the detection of cells apoptosis. SMMC7721 were seeded into a sterile 6-well plate for 24 h. Subsequently, DHTS (1, 2, 4 μg/ml) was added for another 24 h. Then, the cells were fixed in 4% paraformaldehyde solution for 30 min and incubated with Hoechst 33258 dye for 10 min. After washing three times with PBS, apoptotic morphological features were examined and photographed under a fluorescence microscope (OLYMPUS, Japan).
4
Quantitative Analysis of Cartilage ECM Components
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DNA, GAG, and collagen of both adCDMs and pdCDMs were analyzed in this study. adCDMs and pdCDMs were digested at 60 °C overnight in 400 µL and 500 µL papain solution (0.2 M Na2H2PO4, 0.01 M EDTA·2H2O, 250 µg/mL papain, 5 mM L-cysteine, pH 6.0), respectively [22 ]. DNA content of the adCDMs, pdCDMs, and native cartilages was measured using the Hoechst 33,258 assay (C1017, Beyotime) according to the manufacturer’s instructions. This method can detect low amounts of DNA with a limit of detection at the ng level [23 (link)]. A 1,9-dimethylmethylene blue (pH 3.0) assay (HPBIO-JM9048, Hepeng Biology) [24 (link)] was used to measure the sulfated GAG content of the CDM scaffolds, and a hydroxyproline assay (A030-3-1, Njjcbio) was conducted to measure the total collagen content of the dCDMs [25 (link)].
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