Gelatin B (average Mw 20 to 25 kDa, bloom strength 75 g), Poloxamer 188, 25% aqueous glutaraldehyde solution, branched polyethyleneimine 25 kDa (bPEI) and solvents have been purchased from Sigma Aldrich (Steinheim, Germany). Sodium metabisulfite was purchased from Merck (Darmstadt, Germany). Fluorescein isothiocyanate-dextran 150 kDa (FITC-dextran) was derived from TdB (Uppsala, Sweden). RPMI-1640 cell culture medium, HBSS buffer and 4′,6-Diamidino-2-phenylindol solution (DAPI) were obtained from Sigma Aldrich LifeScience GmbH (Seelze, Germany). Fetal calf serum (FCS) was purchased from Lonza, (Basel, Switzerland) and glutamine from Gibco–Thermo Fisher Scientific (Darmstadt, Germany). IbiTreat® microscopy chambers were derived from Ibidi GmbH (Martinsried, Germany). Wheat Germ Agglutinin Alexa Fluor™ 633 conjugate (WGA Alexa Fluor™ 633) was purchased from Thermo Fisher Scientific Inc. (Darmstadt, Germany). Mouse and Human Interleukin 6 (IL-6) as well as Mouse and Human Tumor Necrosis Factor Alpha (TNF-α) ELISA kits were purchased from Shanghai Korain Biotech (Shanghai, China).
Gelatin b
Manufactured by Merck Group
Sourced in United States
Gelatin B is a natural, protein-based material derived from the partial hydrolysis of collagen. It is a versatile lab equipment used for various applications, such as stabilizing emulsions, thickening solutions, and forming gels. Gelatin B possesses specific gelling and viscosity-enhancing properties that make it useful in a range of scientific and industrial applications.
Automatically generated - may contain errors
Lab products found in correlation
Sodium metabisulfite, by Merck Group (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
Poloxamer 188, by Merck Group (1 mentions)
Ibitreat microscopy chamber, by Ibidi (1 mentions)
Fetal calf serum (fcs), by Lonza (1 mentions)
Gelatin a, by Merck Group (1 mentions)
Coomassie blue r250 staining, by Bio-Rad (1 mentions)
Vancomycin hydrochloride, by Merck Group (1 mentions)
Colcemid, by Merck Group (1 mentions)
Glutamine, by Merck Group (1 mentions)
Tris base, by Thermo Fisher Scientific (1 mentions)
Dako fluorescent mounting medium, by Agilent Technologies (1 mentions)
Human recombinant epidermal growth factor, by Thermo Fisher Scientific (1 mentions)
Giemsa stock solution, by Carl Roth (1 mentions)
Dpx mountant, by Merck Group (1 mentions)
Dmem f 12 glutamax, by Thermo Fisher Scientific (1 mentions)
Tween 20, by Merck Group (1 mentions)
Ribonuclease a, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
7 protocols using gelatin b
1
Gelatin-Based Hydrogel Characterization
2
Gelatin Zymography for MMP Quantification
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Mouse aorta was lysed in lysis buffer (Tris 50 mmoL/L, EGTA 0.1 mmoL/L, protease inhibitor cocktail, phosphatase inhibitors cocktail 2 and 3, pH 7.4). Twenty-five μg of proteins were mixed with equal volume of loading buffer (without β-mercaptoethanol) and then heated at 65 °C for 5 min. Equal amount of protein was loaded in 10% SDS-PAGE co-polymerized with 0.5% gelatin B (Sigma-Aldrich, St. Louis, MO, USA). After electrophoresis, the gels were placed in renaturation buffer for 15 min (2×), and equilibrated with developing buffer for 30 min. Then gels were incubated in developing buffer at 37 °C overnight. After staining with coomassie blue solution for 30 min, gels were de-stained with de-staining solution till bands visualized. Densitometric quantification of MMP levels was performed using ImageJ software.
3
Gelatin-based Biomaterials Synthesis
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Gelatin A (from porcine skin, 300 Bloom, isoelectric point (IEP) = 9) and gelatin B (from bovine skin, 225 Bloom, IEP = 5), glutaraldehyde (GA), sodium chloride, Na-salt of polyacrylic acid (MW: 15,000), glycine, were purchased from Sigma-Aldrich. Other chemicals were purchased at Sigma- Aldrich and used as received unless otherwise noted. Deionized dd-water was used to prepare all the solutions.
4
MMP-9 Activation Assay Protocol
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MMP-9 activation assay was performed as previously described with some modifications [10 (link), 14 (link)]. Briefly, 10 ng of recombinant human pro-enzyme MMP-9 (Calbiochem/EMD Millipore) were incubated with recombinant Zmp (100 µg), S. suis or S. pneumoniae (washed bacteria and supernatants as described above) for 1 h at 37 °C. Samples were then separated on 10% zymogram gels containing gelatin B (Sigma) under non-denaturing conditions. Results were visualized after Coomassie Blue R250 staining (Bio-Rad).
5
Hydrogel Synthesis and Characterization
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Gelatin B was obtained from Sigma-Aldrich (Zwijndrecht, the Netherlands) with Bloom number of 225 and an isoelectric point of ~5. A 25 wt% glutaraldehyde solution (in water) was obtained from Acros Organics (Geel, Belgium). DyLight™ 650 NHS Esters and BODIPY-FL®-conjugated vancomycin (designated as fluorescent-labeled vancomycin in this manuscript) were obtained from ThermoFisher Scientific (Bleiswijk, the Netherlands). Vancomycin hydrochloride and all other chemicals used in this study were obtained from Sigma-Aldrich. The zebrafish medium (E3 medium) was prepared according to a standard protocol.42
6
Immunofluorescence-based Cellular Assay
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Dulbecco’s modified Eagle’s medium (DMEM), Phosphate Buffered Saline (PBS), Penicillin/Streptomycin (PS), Glutamine, Insulin, Dexamethasone, sterile water, Gelatin B, AlCl3 hexahydrate, sodium bicarbonate, HCl, KCl, PIPES, Methanol, Acetic acid, DPX mountant, Formaldehyde, DAPI, Tween 20, Triton X-100, Staurosporine, Ribonuclease A, Bovine Serum Albumin (BSA), MMS and Colcemid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Tris Base, DMEM F12 Glutamax and Human recombinant Epidermal Growth Factor (EGF) were from Thermo Fisher Scientific (Waltham, MA, USA). FCS and Horse serum were from BioConcept Ltd (Paradiesrain, Allschwil, Switzerland). Lumogallion (4-Chloro-6-(2,4-dihydroxyphenyl-azo)-1-hydroxybenzene-2-sulfonic acid) was from Santa Cruz Biotechnology Inc (Dallas, TX, USA). DAKO Fluorescent mounting medium was from Agilent Technologies Inc (Santa Clara, CA, USA). Fluoromount G was from SouthernBiotech (Birmingham, AL, USA). Giemsa stock solution was purchased from Carl Roth (Karlsruhe, Germany).
7
Gelatin Zymography for MMP9 Activity
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The supernatants of cell culture were concentrated using centricon (Millipore, 30 kDa cut), and added with non-reducing sample buffer (without DTT). The samples were loaded into the SDS-PAGE gel-containing gelatin B (Sigma-Aldrich). After electrophoresis, gel was washed with 2.5% Triton X-100 in Tris-HCL buffer (pH 7.5). Then the gel was incubated with reaction buffer (15 mM Nacl, 10 mM CaCl2 in Tris-HCl butter (pH 7.5)) overnight at room temperature to induce gelatin lysis by MMP9, and then stained with Coomassie brilliant blue for 30 min followed by destaining.
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