Primary CFs were isolated from the left ventricles of neonatal SD rats following the protocol described in a previous study (12 (link)). After having been harvested from 2-day-old SD rats, the hearts were minced and digested using type II collagenase (120 U/ml; Beijing Solarbio Science and Technology Co., Ltd., Beijing, China). The dissociated cells were collected by centrifugation (200 × g for 8 min at room temperature) and further cultured in MEM medium (Hyclone™) supplemented with fetal bovine serum (FBS; 10%, Hyclone™) (both from GE Healthcare Life Sciences, Shanghai, China) and penicillin-streptomycin antibiotic mix (Sigma-Aldrich; Merck KGaA). Cells were incubated in a humidified incubator under conditions of 95% fresh air and 5% CO2 at 37°C. The adherent cells were cultured to reach over 80% confluence. In excess of 95% of the collected cells were vimentin-positive/α-actin-negative. Cells from passages 2–3 were used. Table II shows the details of grouping, and treatments of the isolated cells.
Penicillin streptomycin antibiotic mix
Manufactured by Merck Group
Sourced in Spain
Penicillin/streptomycin antibiotic mix is a combination of two commonly used antibiotics, penicillin and streptomycin. This product is used in cell culture applications to prevent bacterial contamination.
Automatically generated - may contain errors
Lab products found in correlation
Amphotericin b solution, by Merck Group (2 mentions)
Type 2 collagenase, by Solarbio (1 mentions)
Dulbecco s modified eagle s medium, by Merck Group (1 mentions)
Geneticin, by Merck Group (1 mentions)
Fugene hd transfection reagent, by Promega (1 mentions)
Genipin, by Fujifilm (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (1 mentions)
Hydrochloric acid (hcl), by Merck Group (1 mentions)
Chitosan, by Merck Group (1 mentions)
Co2 incubator, by Eppendorf (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Calcium acetate hydrate, by Merck Group (1 mentions)
Fitc phalloidin, by Merck Group (1 mentions)
Vectashield antifade mounting medium with 4 6 diamidino 2 phenylindole dapi, by Vector Laboratories (1 mentions)
Iron 2 chloride tetrahydrate, by Thermo Fisher Scientific (1 mentions)
Iron 3 chloride hexahydrate, by Thermo Fisher Scientific (1 mentions)
Trypsin egta, by Merck Group (1 mentions)
L glutammine, by Merck Group (1 mentions)
Dulbecco s phosphate buffer saline dpbs, by Merck Group (1 mentions)
6 protocols using penicillin streptomycin antibiotic mix
1
Isolation and Culture of Primary Cardiac Fibroblasts
2
Establishment of Stable Mammalian Cell Lines
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Mammalian cell cultures and transfections followed the methodology described by89 ,90 . Mammalian HEK293 cells (ECACC, UK) were used and they were maintained in complete Dulbecco’s modified Eagle’s medium (DMEM, 4.5 g/l glucose, 110 mg/l sodium pyruvate and L-glutamine, SIGMA-ALDRICH, Spain) supplemented with 10% sterile foetal bovine serum (FBS), 0.1% penicillin: streptomycin antibiotic mix (10,000 U:10 mg/ml, Sigma) and 250 μg /ml sterile filtered 1:100 amphotericin B solution (SIGMA-ALDRICH) in a humid 5% CO2 incubator (HERAEUS, Portugal) at 37 °C. On the day prior to transfection, 2–3 × 105 HEK293 cells were seeded on 6 well plates (SARSTEDT, Portugal) and transfected with Fugene HD transfection reagent (2 DNA: 4 Fugene ratio, PROMEGA) following the manufacturer’s protocol. Seventy-two hours after transfection, 800 μg/ml of the antibiotic Geneticin (SIGMA-ALDRICH) was added to the medium to select and isolate stable receptor cell lines. Cell recovery was monitored daily and the medium was changed every two days until no cell death was observed. Establishment of stable cell lines was confirmed by PCR using specific primers for each receptor.
3
Chitosan-Genipin Hydrogel Synthesis
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Chitosan (CS) at medium molecular weight, 75% deacetylation degree and viscosity 200–800 cps (1 wt % in 1% acetic acid, 25 °C) was purchased from Sigma-Aldrich (prod. N. 448877; CAS 9012-76-4; origin: shrimp shells), as well as acetic acid and ethanol (purity 98%). This CS has been selected among those with low and high molecular weight due to the balance between solubility and capability to react with Genipin, producing hydrogel with modular mechanical properties. Genipin (purity 98% (HPLC)) was purchased from Wako chemicals USA (Richmond, VA, USA) (prod. N. 078-03021). All the products were used without any further purification. Eagle’s minimum essential medium (E-MEM), Roswell Park Memorial Institute medium (RPMI-1640), fetal bovine serum (FBS), L-glutammine, penicillin/streptomycin antibiotic mix, Dulbecco’s phosphate buffer saline (D-PBS), paraformaldehyde (PFA) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich. All reagents, medium supplements and plastic-ware were purchased as the cell-culture was tested. Phorbol-12-myristate-13-acetate (PMA; prod. n. P8139; CAS 0016561298), propanol and HCl were purchased form Sigma-Aldrich.
4
Culturing Primary Human Dermal Fibroblasts
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Primary normal human dermal fibroblasts (NHDF) isolated from human adult skin were purchased from Lonza Clonetics TM (Lonza, Walkersville, USA) [23] (link). Cells were cultured and maintained following the manufactures instructions and were grown in a humid 5 % CO2 incubator (Eppendorf, Hamburg, Germany) at 37°C. Cell growth was performed in Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich, Taufkirchen, Germany ) with 4.5 g/l glucose, 110 mg/l sodium pyruvate and L-glutamine supplemented with 0.1 % penicillin/streptomycin antibiotic mix (10.000 U:10 mg/ml, Sigma-Aldrich, Taufkirchen, Germany), 250 μg/ml sterile filtered 1:100 amphotericin B solution (Sigma-Aldrich, Taufkirchen, Germany) and 10% FBS (Sigma-Aldrich, Taufkirchen, Germany). For the in vitro scratch assays, the cells were maintained in Dulbecco's modified Eagle's medium free of FBS for 2 days before the experiments, in order to obtain arrest of proliferation.
5
Synthesis and Characterization of DM/n-HA Nanostructures
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For DM and DM/n-HA nanostructures synthesis, iron (II) chloride tetrahydrate (FeCl2·4H2O, ≥99%), iron (III) chloride hexahydrate (FeCl3·6H2O, ≥97%) and Dextran from LeuconostocMesenteroids (Mw 6000 Da) were purchased by Alfa Aesar. Sodium hydroxide anhydrous pellets (NaCl, ≥99%), hydrochloric acid (≥37% wt. in water), phosphoric acid (≥85% wt. in water), ammonium hydroxide ((NH4)OH, ≥30% wt. in water), and calcium acetate hydrate (Ca(CH3COO)2·XH2O, ≥99%) were purchased from Sigma–Aldrich. Ultrapure water (18.2 MΩ/cm, obtained by a Milli-Q® Direct Water Purification System, Merck Millipore, Darmstadt, Germany) has been used in all the experiments.
For the cell-based experiments, Eagle’s Minimum Essential Medium (E-MEM), fetal bovine serum (FBS), L-glutammine, penicillin/streptomycin antibiotic mix, Dulbecco’s phosphate buffer saline (D-PBS), trypsin-EGTA, paraformaldehyde (PFA), Triton X-100, as well as the fluorescent dye phalloidin-FITC were all purchased from Sigma–Aldrich (Milan, Italy). The Vectashield Anti-fade Mounting Medium with 4′,6-diamidino-2-phenylindole DAPI (Vector Laboratories, Peterborough, UK) was used for nuclear staining. All reagents, media supplements, and plastic-ware were supplied as cell-culture tested.
For the cell-based experiments, Eagle’s Minimum Essential Medium (E-MEM), fetal bovine serum (FBS), L-glutammine, penicillin/streptomycin antibiotic mix, Dulbecco’s phosphate buffer saline (D-PBS), trypsin-EGTA, paraformaldehyde (PFA), Triton X-100, as well as the fluorescent dye phalloidin-FITC were all purchased from Sigma–Aldrich (Milan, Italy). The Vectashield Anti-fade Mounting Medium with 4′,6-diamidino-2-phenylindole DAPI (Vector Laboratories, Peterborough, UK) was used for nuclear staining. All reagents, media supplements, and plastic-ware were supplied as cell-culture tested.
6
Transient Transfection of HEK293 Cells
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HEK293 cells (ECACC collection, UK) were maintained in complete Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich, Spain) with 4.5 g/L glucose, 110 mg/L sodium pyruvate and L-glutamine supplemented with 10 % sterile foetal bovine serum and 0.1 % penicillin: streptomycin antibiotic mix (10.000 U:10 mg/ml, Sigma) and 250 μg/ml sterile filtered 1:100 amphotericin B solution (Sigma, Spain) in a humid 5 % CO2 incubator (Heraeus, Portugal) at 37 °C. One day prior to transfection, 2-3 x 10 5 cells were seeded into 6 well plates (Sarstedt, Portugal) and cells were transiently transfected using Fugene 6 transfection reagent (1 : 6 DNA : Fugene, Roche, Germany) following the manufacturer's protocol. Simultaneous transient transfections of a vector expressing green fluorescent protein were performed to estimate the efficiency and success of cell transfections. The capacity of the PACAP peptides PACAP-27 and hPACAP-38 to activate the teleost receptor cAMP-signalling pathway was assayed 72 hours after cell transfections. HEK293 stable cell lines expressing human RAMP1 were generated to assess the effect of transmembrane accessory proteins on the pharmacology of PACAP receptor function.
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