To determine non-cellular hydroxyapatite deposition leading to changes in calcium concentration in the medium, PLGA ± aCaP discs (3D meshes and 2D films, respectively) were incubated in 2 mL DMEM with 10% of FBS and Antibiotic-Antimycotic 1X (Thermo Fisher Scientific, Basel, Switzerland) for two weeks at 37 °C and 5% CO2, changing the medium every 3 or 4 days. A control was included using the medium without discs. Sample size was n = 3. The calcium concentration remaining in the solution of the samples was measured by a colorimetric assay kit (Sigma-Aldrich, Zurich, Switzerland) using a microplate reader (Tecan, Spark, Männedorf, Switzerland).
Antibiotic antimycotic 1x
Manufactured by Thermo Fisher Scientific
Sourced in Switzerland
Antibiotic-Antimycotic (1X) is a sterile solution containing antibiotics and antimycotics designed for use in cell culture applications. The solution is formulated to inhibit the growth of bacteria, fungi, and yeasts.
Automatically generated - may contain errors
Lab products found in correlation
Microplate reader, by Tecan (1 mentions)
Colorimetric assay kit, by Merck Group (1 mentions)
Lookout mycoplasma pcr detection kit, by Merck Group (1 mentions)
Collagenase type 4, by Worthington (1 mentions)
Dnase, by Merck Group (1 mentions)
Dispase, by Merck Group (1 mentions)
D45131vl, by Thermo Fisher Scientific (1 mentions)
Mycoplasma removal agent, by Bio-Rad (1 mentions)
Recombinant human il 1β, by R&D Systems (1 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Roche (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Collagenase type 4, by Merck Group (1 mentions)
Optiprep, by Merck Group (1 mentions)
100 μm cell strainer, by BD (1 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (1 mentions)
8 protocols using antibiotic antimycotic 1x
1
Hydroxyapatite Deposition in PLGA Scaffolds
2
Isolation and Characterization of Murine Heart Fibroblasts
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Fibroblasts were isolated from young (3–4 months old) and old (24–30 months old) male and female mouse hearts. The isolation method was performed, as described below (see STAR methods details). Cells were cultured in complete growth medium [DMEM/F-12 1:1, 10% fetal bovine serum, Antibiotic/Antimycotic 1X (ThermoFisher # 15240062)] in a 5% CO2 wet incubator at 37°C. The cultures are regularly tested for mycoplasma contamination using the LookOut Mycoplasma PCR Detection Kit (Millipore-Sigma # MP0035), and the results were negative. Cells prepared from old animals were also carefully checked for phenotype changing.
A unique identification number is provided to each cell line prepared, based on the following nomenclature “Mouse age (in months-old). Mouse ID_Sex”. For instance, 30.80_M refers to the cells from the 30 months-old mouse #80, which is a male. Fibroblasts are stored in a dedicated storage place in a liquid nitrogen tank, and the location of each vial is recorded in a folder.
A unique identification number is provided to each cell line prepared, based on the following nomenclature “Mouse age (in months-old). Mouse ID_Sex”. For instance, 30.80_M refers to the cells from the 30 months-old mouse #80, which is a male. Fibroblasts are stored in a dedicated storage place in a liquid nitrogen tank, and the location of each vial is recorded in a folder.
3
Cardiac Fibroblast Isolation and Culture
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Cardiac fibroblasts were prepared as previously described.38 (link) Briefly, the slice of the myocardium (mid-myocardium to apical) was cut into 1 mm3 –sized pieces and incubated in a digestion buffer containing 2 mg/mL Collagenase type IV (Worthington Cat#LS004188), 2 mg/mL Dispase (Sigma Cat# D4818-2MG) and DNAse (14.8 U/mL, prepared from Millipore-Sigma #D45131VL) in PBS Ca2+/Mg2+ (ThermoFisher #14190144). The supernatant was passed through a Falcon 40 μm strainer (Fisher-Scientific #08-771-1) and mixed with an ice-cold quenching buffer [2% fetal bovine serum (from HyClone #SH30071.03), 14.8 U/mL DNAse (prepared from Millipore-Sigma #D45131VL) in PBS without Ca2+/Mg2+ (ThermoFisher #14190250)]. The final cell suspension was spun down at 300 g to pellet the cells, washed once with sterile D-PBS, then resuspended in the complete growth medium [DMEM/F-12 1:1, 10% fetal bovine serum, Antibiotic/Antimycotic 1X (ThermoFisher # 15240062)]. After isolation, cells were left seeding undisturbed for 48 h, then were thoroughly washed twice with sterile D-PBS.
To prevent mycoplasma contamination, medium was supplemented with 0.5 μg/mL of Mycoplasma removal agent (Biorad #BUF035) for the first 2 weeks following the isolation.
To prevent mycoplasma contamination, medium was supplemented with 0.5 μg/mL of Mycoplasma removal agent (Biorad #BUF035) for the first 2 weeks following the isolation.
4
Hypoxia-Induced JAR Secretome Analysis
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On day 5 of JAR cell treatment with CoCl2 (versus control condition), media exchange was performed to apply serum-free media for both conditions. 24 hr later, conditioned media samples were collected and applied to 10 kDa molecular weight cutoff filters (Amicon Ultra), with centrifugation for 20 min at 4000 × g, 4°C. Filters were washed with one volume of hTERT-HM base medium (see below), with repeat centrifugation. Concentrated proteins retained in suspension above the filter were collected and stored at –80°C.
hTERT-HM cells (generously supplied by Dr. Jennifer Condon, Wayne State; STR reference profile not available) were grown in DMEM/F12 medium (Gibco) with 10% FBS and Antibiotic-Antimycotic (1X, Gibco). Cells were stimulated by addition of recombinant human IL-1β (R&D Systems) and/or filter-concentrated JAR cell conditioned media as indicated, 6 hr prior to collection of cellular RNA.
hTERT-HM cells (generously supplied by Dr. Jennifer Condon, Wayne State; STR reference profile not available) were grown in DMEM/F12 medium (Gibco) with 10% FBS and Antibiotic-Antimycotic (1X, Gibco). Cells were stimulated by addition of recombinant human IL-1β (R&D Systems) and/or filter-concentrated JAR cell conditioned media as indicated, 6 hr prior to collection of cellular RNA.
5
Genetically Engineered Mouse Glioma Models
6
Antimicrobial Activity Screening Protocol
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The antimicrobial effect of the secretion was performed following Lu et al.'s [21 (link)] protocol, which was only carried out with samples from the Punucapa locality due to sample availability. Standard bacterial strains used in antimicrobial assays were the Gram-positive bacterium Staphylococcus aureus (ATCC 25923) and the Gram-negative bacterium Escherichia coli (ATCC 25922). Bacteria were first grown in LB (Luria–Bertani) broth to an OD600 nm of 0.8. An aliquot (10 µL) of the bacterial suspension was added to 8 mL of the fresh LB broth with 0.7% agar and poured over a 90 mm Petri dish containing 25 mL of 1.5% agar in LB broth. After the top agar hardened, a 20 μL aliquot of the test sample filtered on a 0.22 μm millipore filter was dropped onto the surface of the top agar and completely dried before being incubated overnight at 37°C. If a secretion displayed antimicrobial activity, a clear zone would form on the surface of the top agar, indicating the inhibition of the bacterial growth. Antibiotic-antimycotic 1X (Gibco™) was used as the positive control, and acetic acid (5%) was used as the negative control.
7
Isolation and Culture of Human Hepatic Stellate Cells
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Human HSC (hHSC) were isolated from wedge sections of human liver tissue, obtained from patients undergoing liver surgery at the Royal Free Hospital after giving informed consent (NC2015.020 (B-ERC-RF)), as described before (38, 49) . Briefly, 10g of total human liver tissue was digested with 0.01% Collagenase type IV (Sigma Aldrich), 0.05% Pronase (Calbiochem) and 0.001% DNase I (Sigma Aldrich). The homogenate was filtered through a 100μm cell strainer (BD Falcon) and the flow-through was centrifuged at 50 x g for 2 minutes at 4°C to remove hepatocytes. After washing the supernatant, gradient centrifugation was performed at 1400 x g for 17 minutes using a 11.5% Optiprep gradient (Sigma Aldrich). Finally, the interface was collected and washed. The obtained hHSC were cultured in Iscove's Modified Dulbecco's Medium supplemented with 20% foetal bovine serum (FBS), 2 mM/l glutamine, nonessential amino acids 1x, 1.0 mM/l sodium pyruvate, antibiotic-antimycotic 1x (all GIBCO), referred to as complete HSC medium hereinafter.
Experiments were performed on cells between passage 3 and 8 employing at least three different cell preparations/donors for all experiments.
Experiments were performed on cells between passage 3 and 8 employing at least three different cell preparations/donors for all experiments.
8
SV40 Large T Antigen Immortalized Mouse Fibroblasts
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Simian virus 40 large T antigen immortalized mouse embryonic fibroblasts (MEFs) were kindly provided by Dr Benoit Viollet. AMPKα1/α2 -/-and wt MEFs were obtained from 10.5 day postcoitum embryos, the genotype was confirmed by PCR and immunoblot analysis.
MEFs from the second or third culture passage were immortalized by introducing the SV 40 large T antigen using the pSV-Ori-vector (30) . MEFs were cultured in Dulbecco's Modified Eagle Medium supplemented with 10% FBS, 1mM sodium pyruvate and Antibiotic-Antimycotic 1x (all GIBCO).
MEFs from the second or third culture passage were immortalized by introducing the SV 40 large T antigen using the pSV-Ori-vector (30) . MEFs were cultured in Dulbecco's Modified Eagle Medium supplemented with 10% FBS, 1mM sodium pyruvate and Antibiotic-Antimycotic 1x (all GIBCO).
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