Chondrocyte attachment onto CS/Gel/PVA hydrogels was visualized using scanning electron microscopy (SEM) (16538, Electron Microscopy Science, Hatfield, PA, USA). Constructs (5 × 105 cells) were fixed with 2.5% v/v glutaraldehyde (Sigma-Aldrich) in 0.1 M sodium cacodylate (Sigma-Aldrich) at pH 7.2 for 24 h. Samples were washed for 5 min in 0.1 M PBS at pH 7.4 and further dehydrated for 10 min in a serially diluted ethanol solution, ranging from 30% to 99.99%, for 30 min each wash (E7148, Sigma-Aldrich, Merck, Darmstadt, Germany). All specimens were dried with a critical point dryer CO2 chamber (K850, Quorum Technologies, Kent, UK). Images were acquired with a FIB-SEM Crossbeam 550 field emission microscope (Carl Zeiss, Oberkochen, Baden-Württemberg, Germany) at 5 kV.
E7148
Manufactured by Merck Group
Sourced in Germany
The E7148 is a piece of laboratory equipment manufactured by Merck Group. It is designed for general laboratory use. The core function of the E7148 is to provide a controlled environment for various scientific experiments and analysis. Further details on the intended use or specific features of this product are not available.
Automatically generated - may contain errors
Lab products found in correlation
Fib sem crossbeam 550 field emission microscope, by Zeiss (4 mentions)
Carbon based electrically conductive carbon double sided adhesive discs, by Agar Scientific (2 mentions)
Agb7340, by Agar Scientific (2 mentions)
Glutaraldehyde, by Merck Group (1 mentions)
Sodium cacodylate, by Merck Group (1 mentions)
H 2535, by Bachem (1 mentions)
P1675, by Merck Group (1 mentions)
Mirneasy micro kit, by Qiagen (1 mentions)
Chloroform, by Merck Group (1 mentions)
Rnase free dnase set, by Qiagen (1 mentions)
6 protocols using e7148
1
Chondrocyte Attachment on Hydrogel Scaffolds
2
DAMGO, Ethanol, and GABAA Antagonist Interactions
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We used the MOR agonist [D-Ala2 (link), NMe-Phe4 (link), Gly-ol5 (link)]-enkephalin (DAMGO; H-2535, Bachem), ethanol (ethanol, E7148, Sigma-Aldrich) and GABAA receptor antagonist picrotoxin (PTX, P1675, Sigma-Aldrich). Other reagents used for making solutions were purchased from Sigma-Aldrich or Fisher Scientific.
3
Hydrogel Microarchitecture and Cell Adhesion
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The microarchitecture and topology of the Gel/CS/PVA hydrogel; as well as the cell adherence and morphology in chondrogenic constructs, were observed by SEM. Briefly, all samples were fixed in 3% (v/v) glutaraldehyde in a buffering solution of 0.1 M sodium cacodylate (pH 7.2) for 48 h (16538, Electron Microscopy Science, Hatfield, PA, USA). Subsequently, the samples were dehydrated in ethanol ranging from 30% to 99.99% for 30 min each (E7148, Sigma-Aldrich, Merck, Darmstadt, Germany). Then, they were dried using a critical point dryer CO2 chamber (K850, Quorum Technologies, Lewes, UK), as previously reported. Finally, all dried samples were mounted in 25 mm diameter aluminum pin stubs and mounted on them using 25-mm-diameter carbon-based electrically conductive carbon double-sided adhesive discs (Agar Scientific, Stansted, UK). The samples were coated with gold in two thin layers in two cycles of 20 sec each by plasma-assisted deposition using a manual sputter coater (AGB7340, Agar Scientific, Stansted, UK). The images were acquired with a FIB-SEM Crossbeam 550 field emission microscope Zeiss (Oberkochen, Baden-Württemberg, Germany) at 4 kV.
4
Microarchitecture Characterization of Native and Engineered Extracellular Matrices
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The microarchitecture of the native samples and dECM-BD was observed by SEM. Briefly, all samples were fixed in 3% (v/v) glutaraldehyde in a buffering solution of 0.1M sodium cacodylate (pH 7.2) for 48 h, (16538, Electron Microscopy Science, Hatfield, PA, USA). Subsequently, all samples were dehydrated at various ethanol concentrations, ranging from 30% to 99.99%, 30 min each, (E7148, Sigma-Aldrich, Merck, Darmstadt, Germany) Finally, they were dried using a critical point dryer CO2 chamber (K850, Quorum Technologies, Kent, UK), as previously reported [18 (link),19 (link)]. The images were acquired with a FIB-SEM Crossbeam 550 field emission microscope (Zeiss Oberkochen, Germany) at 5 kV.
5
Modified miRNeasy Micro Kit Protocol
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Minor changes have been introduced to manufacturer's protocol for miRNeasy Micro Kit (Qiagen, Limburg, the Netherlands). Briefly, 7 µl of BME (14.3 M solution) was added to each 700 µl QIAzol solution prior use with HFDtablet samples. Incubation was performed for 20 min at RT, followed by the addition of 140 µl of chloroform (biotech grade, 496189, Sigma-Aldrich). After additional 10 min of incubation, the samples entered the phase separation step with extension of total centrifugation time from 15 to 30 min. The upper phases were collected and mixed with 1.5 volume of pure ethanol (190 proof, molecular biology, E7148, Sigma-Aldrich). Next, samples were placed on column and washed according to original protocol including DNase treatment on column (performed using RNase-free DNase Set (Qiagen)). Thereafter, RNA was eluted with 25 µl of RNase-free water.
6
Hydrogel Microarchitecture and Cell Adhesion
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The microarchitecture and topology of the Gel/CS/PVA hydrogel; as well as the cell adherence and morphology in chondrogenic constructs, were observed by SEM. Briefly, all samples were fixed in 3% (v/v) glutaraldehyde in a buffering solution of 0.1 M sodium cacodylate (pH 7.2) for 48 h (16538, Electron Microscopy Science, Hatfield, PA, USA). Subsequently, the samples were dehydrated in ethanol ranging from 30% to 99.99% for 30 min each (E7148, Sigma-Aldrich, Merck, Darmstadt, Germany). Then, they were dried using a critical point dryer CO2 chamber (K850, Quorum Technologies, Lewes, UK), as previously reported. Finally, all dried samples were mounted in 25 mm diameter aluminum pin stubs and mounted on them using 25-mm-diameter carbon-based electrically conductive carbon double-sided adhesive discs (Agar Scientific, Stansted, UK). The samples were coated with gold in two thin layers in two cycles of 20 sec each by plasma-assisted deposition using a manual sputter coater (AGB7340, Agar Scientific, Stansted, UK). The images were acquired with a FIB-SEM Crossbeam 550 field emission microscope Zeiss (Oberkochen, Baden-Württemberg, Germany) at 4 kV.
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