Serum-free cell culture media obtained from cells cultured in 2D monolayers and 3D spheroids (two independent copies of the same cell line grown in 2D or 3D) were mixed 3:1 with a sample buffer (containing 10% SDS). Samples (5 μg of total proteins) were run at 4 °C under non-reducing conditions on 10% SDS-PAGE co-polymerized with 1 mg/mL of type I gelatin. After electrophoresis, the gels were washed in 2.5% Triton X-100 (2 washes for 30 min each), and incubated overnight in an incubation buffer (Tris-HCl 50 mM, CaCl2 5 mM, 0.02% NaN3, pH 7.5) at 37 °C. Gels were stained with Coomassie brilliant blue R250 to reveal MMPs’ gelatinolytic activity which appeared as clear bands on a blue background, and were quantified by densitometric analysis (UVBand, Eppendorf, Milan, Italy). Each experiment was repeated three times.
Uvband
Manufactured by Eppendorf
Sourced in Italy
The UVBand is a compact and versatile ultraviolet transilluminator designed for the visualization and documentation of nucleic acid samples separated by gel electrophoresis. It features a UV-transparent glass surface and a built-in UV light source.
Automatically generated - may contain errors
9 protocols using uvband
1
Gelatin Zymography Assay for MMPs
2
Gelatin Zymography Assay for MMPs
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Serum-free culture media were mixed 3:1 with sample buffer (containing 10% SDS). Samples (5 μg of total protein per sample) were run under non-reducing conditions without heat denaturation on 10% polyacrylamide gel (SDS-PAGE) co-polymerized with 1 mg/mL of type I gelatin. The gels were run at 4 °C. After SDS-PAGE, the gels were washed twice in 2.5% Triton X-100 for 30 min each, and incubated overnight in a substrate buffer at 37 °C (Tris-HCl 50 mM, CaCl2 5 mM, 0.02% NaN3, pH 7.5). MMP gelatinolytic activity, detected after staining the gels with Coomassie brilliant blue R250 as clear bands on a blue background, was quantified by densitometric scanning (UVBand, Eppendorf, Milan, Italy).
3
Quantitative Analysis of Collagen I and MMP-1
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Collagen type I (COL-I) and matrix metalloproteinase (MMP)-1 protein levels secreted by tenocytes in serum-free cell supernatants were analyzed by slot blot analysis, as previously detailed [18 (link)]. Membranes were incubated for 1 h at room temperature with primary monoclonal antibodies to COL-I (1:1000 in TBST) (Sigma-Aldrich, Milan, Italy) or MMP-1 (1 µg/mL in TBST) (Millipore, Milan, Italy). Immunoreactive bands were revealed by the Amplified Opti-4CN substrate (Amplified Opti-4CN, Bio Rad, Segrate, Milan, Italy) and quantification was obtained after densitometric scanning of immunoreactive bands (UVBand, Eppendorf, Italy).
4
Slot Blot Analysis of Tenocyte Secretions
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Collagen type I and III (COL-I, COL-III), matrix metalloproteinase (MMP)-1 protein levels secreted by tenocytes were assessed in duplicate samples by Slot blot in serum free cell culture medium, as previously detailed [21 ]. Membranes were incubated for 1 h at room temperature in monoclonal antibody to COL-I (1:1000 in TBST) (Sigma-Aldrich, Milan, Italy), COL-III (1:2000 in TBST) (Sigma-Aldrich, Milan, Italy), MMP-1 (1 µg/mL in TBST) (Millipore, Milan, Italy). Immunoreactive bands, revealed by the Amplified Opti-4CN substrate (Amplified Opti-4CN, Bio Rad, Italy), were scanned densitometrically (UVBand, Eppendorf, Italy).
5
Quantifying Tenocyte MMP-2 Gelatinolytic Activity
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MMP-2 levels and activity were analyzed in serum-free culture supernatants (5 μg of total protein per sample) in tenocytes cultured for 48 h by SDS-zymography on 10% polyacrylamide gels co-polymerized with 1 mg/mL type I gelatin. The gels were run at 4 °C and, after SDS-PAGE, they were washed twice in 2.5% Triton X-100 for 30 min each and incubated overnight in a substrate buffer at 37 °C (Tris-HCl 50 mM, CaCl2 5 mM, NaN3 0.02%, pH 7.5). After staining and destaining the gels, MMP gelatinolytic activity was detected as clear bands on a blue background after staining the gels with Coomassie brilliant blue R250. Clear bands were quantified by densitometric scanning (UVBand, Eppendorf, Italy).
6
Quantifying Collagen and MMP-1 in Fibroblasts
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Collagen type I and III (COL-I and COL-III) and matrix metalloproteinase (MMP)-1 protein levels secreted by muscle fibroblasts were analysed by Slot Blot in serum-free cell supernatants as previously described [67 (link)]. After blocking, membranes were incubated for 1 h at room temperature with primary polyclonal antibodies to COL-I (1:1000 in 1X Tris-Buffered Saline plus 0.1% Tween 20, TBST) (Sigma-Aldrich, Milan, Italy), COL-III (1:1000 in TBST) (Sigma-Aldrich), or MMP-1 (1 μg/mL in TBST) (Millipore, Milan, Italy). After 1 h incubation with HRP-conjugated antibody (1:20,000 in TBST), immunoreactive bands were revealed by the Amplified Opti-4CN substrate (Amplified Opti-4CN, Bio Rad, Segrate, Milan, Italy) and quantified by densitometric scanning (UVBand, Eppendorf, Milan, Italy).
7
Quantification of MMP Gelatinolytic Activity
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Serum-free cell culture supernatants (5 μg of total protein per sample) were run on 10% polyacrylamide gels co-polymerised with 1 mg/mL type I gelatine. The gels were run at 4 °C and, after Sodium Dodecyl Sulphate—Polyacrylamide gel electrophoresis (SDS-PAGE), they were washed twice in 2.5% Triton X-100 for 30 min each to remove SDS and incubated overnight in a substrate buffer at 37 °C (50 mM Tris-HCl, 5 mM CaCl2, 0.02% NaN3, pH 7.5). After staining and destaining the gels, MMP gelatinolytic activity was detected as clear bands on a blue background. Clear bands on the zymogram were quantified by densitometric scanning (UVBand, Eppendorf, Milan, Italy).
8
Quantifying MMP-2 Gelatinolytic Activity
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SDS-zymography was used to analyze MMP-2 activity of secreted protein in cell culture medium, as previously described [21 ]. MMP gelatinolytic activity, detected after staining the gels with Coomassie brilliant blue R250 as clear bands on a blue background, were quantified by densitometric scanning (UVBand, Eppendorf, Italy).
9
Gelatin Zymography for MMP Activity
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Serum-free culture media were mixed 3:1 with sample buffer (containing 10% SDS). Samples (5 μg of total protein per sample) were run under non-reducing conditions without heat denaturation on 10% polyacrylamide gel (SDS-PAGE) co-polymerized with 1 mg/mL of type I gelatin. The gels were run at 4°C. After SDS-PAGE, the gels were washed twice in 2.5% Triton X-100 for 30 min each, and incubated overnight in a substrate buffer at 37 °C (Tris-HCl 50 mM, CaCl2 5 mM, 0.02% NaN3, pH 7.5). MMP gelatinolytic activity, detected after staining the gels with Coomassie brilliant blue R250 as clear bands on a blue background, was quantified by densitometric scanning (UVBand, Eppendorf, Milan, Italy).
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