MC3T3-E1 cells were seeded in 12 multi well culture dishes at a density of 10,000/cm2 and transfected with Suv420h2 specific or scrambled siRNA as described above. On the next day, the medium was changed and cell differentiation was induced. After ten days, cell number and ALP-activity were analyzed. For determination of cell number (DNA amount employed as surrogate), cell layers were washed with PBS and frozen with 1 mM Tris–HCl buffer (pH 8.0) containing 0.1 mM EDTA. During thawing, Hoechst 33258 dye (Polysciences, Warrington, PA) was added (1 μg/mL) and, after an incubation of 15 min at room temperature, the fluorescence was measured (excitation 360, emission 465 nm). The amount of DNA was estimated using a standard curve prepared from calf thymus DNA (Roche). Thereafter, alkaline phosphatase (ALP) activity was measured with p-nitrophenylphosphate (Sigma) (2.5 mg/mL in 0.1 M diethanolamine buffer [pH 10.5], 150 mM NaCl, 2 mM MgCl2) by incubation of the cell layers for 20 min at room temperature. Absorption was measured in a microplate reader at 405/490 nm. ALP activity per well was estimated using a standard curve prepared from calf intestinal ALP (Roche) and referred to the amount of DNA previously measured.
Hoechst 33258 dye
Manufactured by Polysciences
Sourced in United Kingdom
Hoechst 33258 dye is a fluorescent stain used for the detection and quantification of DNA in biological samples. It binds to the minor groove of DNA, resulting in a fluorescent signal that can be measured using appropriate instrumentation.
Automatically generated - may contain errors
Lab products found in correlation
Calf thymus dna, by Roche (2 mentions)
Calf intestinal alp, by Roche (2 mentions)
P nitrophenyl phosphate, by Merck Group (1 mentions)
Papain, by Polysciences (1 mentions)
Calf thymus dna, by Merck Group (1 mentions)
Chondroitin sulfate, by Merck Group (1 mentions)
Papain, by Merck Group (1 mentions)
L hydroxyproline, by Merck Group (1 mentions)
Ethidium homodimer 1, by Biotium (1 mentions)
Microplate reader, by Tecan (1 mentions)
Proteinase k, by Roche (1 mentions)
5 protocols using hoechst 33258 dye
1
Quantification of Osteoblast Differentiation
2
Cartilage Extracellular Matrix Quantification
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The in vitro–formed cartilage and the tissue bridge were harvested separately and
each digested in 40 µg/mL papain (Sigma-Aldrich). The native cartilage removed
from the bone was digested in 80 µg/mL papain for 48 hours at 65°C as previously described.34 (link)
The DNA content of the papain digests was quantified using a fluorometric assay
(excitation, 356 nm; emission, 458 nm) and Hoechst 33258 dye (Polysciences) and
compared with a standard curve generated using serial dilutions of calf thymus
DNA (Sigma-Aldrich) as previously described.34 (link)
To quantify collagen content, papain digests were acid hydrolyzed for 18 hours at
110°C. Hydroxyproline content was measured using Chloramine-T/Ehrlich’s reagent
assay and spectrophotometry (λ = 560 nm) as previously described.34 (link)
A standard curve was generated with L-hydroxyproline (Sigma-Aldrich).
Sulfated glycosaminoglycan content in the papain digests was quantified using
dimethylmethylene blue dye and spectrophotometry (λ = 525 nm) and compared with
a standard curve generated using chondroitin sulfate (Sigma-Aldrich) as
previously described.34 (link)
each digested in 40 µg/mL papain (Sigma-Aldrich). The native cartilage removed
from the bone was digested in 80 µg/mL papain for 48 hours at 65°C as previously described.34 (link)
The DNA content of the papain digests was quantified using a fluorometric assay
(excitation, 356 nm; emission, 458 nm) and Hoechst 33258 dye (Polysciences) and
compared with a standard curve generated using serial dilutions of calf thymus
DNA (Sigma-Aldrich) as previously described.34 (link)
To quantify collagen content, papain digests were acid hydrolyzed for 18 hours at
110°C. Hydroxyproline content was measured using Chloramine-T/Ehrlich’s reagent
assay and spectrophotometry (λ = 560 nm) as previously described.34 (link)
A standard curve was generated with L-hydroxyproline (Sigma-Aldrich).
Sulfated glycosaminoglycan content in the papain digests was quantified using
dimethylmethylene blue dye and spectrophotometry (λ = 525 nm) and compared with
a standard curve generated using chondroitin sulfate (Sigma-Aldrich) as
previously described.34 (link)
3
Cell Viability Analysis in NP Tissue
4
Quantification of Alkaline Phosphatase Activity
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ALPL activity was determined in relation to total DNA present in samples as a surrogate for cell number. Cells were washed with PBS and frozen in 1 mM Tris–HCl buffer (pH 8.0) containing 0.1 mM EDTA. During thawing, DNA content was determined using 1 μg/ml Hoechst 33258 dye (Polysciences, Warrington, PA). After incubation of 15 min at room temperature, the fluorescence intensity was measured (excitation 360, emission 465 nm). The amount of DNA was estimated using a standard curve prepared from calf thymus DNA (Roche). ALPL activity was quantified with p-nitrophenylphosphate (2.5 mg/ml in 0.1 M diethanolamine buffer [pH 10.5], 150 mM NaCl, 2 mM MgCl2) by incubation of the cells for 15 min at room temperature and measurements were referenced to a standard curve prepared from calf intestinal ALP (Roche). Absorption was measured in a microplate reader at 405/490 nm (Tecan) and the instrument’s software Magellan version 7.2. ALPL activity was expressed as arbitrary units per milligram DNA and referenced to fold change to control.
5
Quantifying GAG Production from Cell Scaffolds
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For biochemical analysis, one half scaffold was digested in 0.5 mg/ml Proteinase K (Roche, Basel, Switzerland) at 56°C for 16 h. Proteinase K (PK) was then deactivated with a 10 min incubation at 96°C, and the samples stored at -20°C for analysis. Total amount of glycosaminoglycans (GAGs) produced by cells, measured in both collected media and PK digests, was determined using the 1.9-dimethyl methylene blue (DMMB) assay and normalised to the DNA content of each PK digest measured using Hoechst 33,258 dye (Polysciences Inc., Warrington, United States).
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