MSC were obtained from subcutaneous adipose tissue from 13 healthy human donors that became available during the living kidney donation procedure. All donors provided written informed consent as approved by the Medical Ethical Committee of the Erasmus University Medical Center Rotterdam (protocol no. MEC-2006-190). MSC were isolated and phenotypically characterized by the expression of CD13, CD73, CD90, and CD105 and the absence of CD31 and CD45 as described previously (16 (link)). MSC were cultured in minimum essential medium-α (MEM-α) (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 100 IU/ml penicillin, 100 mg/ml streptomycin (P/S), 2 mM L-glutamine, and 15% fetal bovine serum (FBS) (all Lonza, Verviers, Belgium). Cultures were kept at 37°C, 5% CO2, and 95% humidity. At 90% confluence, adherent cells were collected from culture flasks by incubation in 0.05% trypsin-EDTA (Life Technologies, Bleiswijk, The Netherlands) at 37°C. MSC between passages 2 and 6 were used for MP generation.
Streptomycin p s
Manufactured by Lonza
Sourced in United Kingdom
Streptomycin (P/S) is a laboratory equipment product manufactured by Lonza. It is an antimicrobial agent used in cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Lonza (8 mentions)
L glutamine, by Lonza (3 mentions)
Heracell 150i, by Thermo Fisher Scientific (3 mentions)
α mem, by Merck Group (2 mentions)
Trypsin edta, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Lonza (2 mentions)
Tissue culture flasks, by Sarstedt (2 mentions)
Iscove modified dulbecco media (imdm), by Lonza (2 mentions)
A2780, by European Collection of Cell Cultures (2 mentions)
Skov3, by European Collection of Cell Cultures (2 mentions)
Mccoy s 5a medium modified, by Lonza (2 mentions)
Aspc 1, by American Type Culture Collection (2 mentions)
Mia paca 2, by American Type Culture Collection (2 mentions)
Roswell park memorial institute (rpmi), by Lonza (2 mentions)
Resazurin based in vitro toxicology assay kit, by Merck Group (1 mentions)
Alamar blue, by Thermo Fisher Scientific (1 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Methanol, by Merck Group (1 mentions)
175 cm2 culture flasks, by Greiner (1 mentions)
9 protocols using streptomycin p s
1
Isolation and Characterization of Adipose-Derived MSCs
2
Cytotoxicity Assessment of ZEA Compound
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Standard of ZEA (Z2125-10MG, Sigma) was obtained from Sigma-Aldrich (Sintra, PT). Ethanol was obtained from Panreac (Barcelona, ES) and methanol from Merck (Lisbon, PT). Fetal bovine serum (FBS), cell culture EMEM (Eagle’s Minimum Essential Medium), ultraglutamine 1 (L-Gln) (200 μmol L−1), non-essential amino acids (NEAA) 100X, Trypsin-Ethylenediamine tetraacetic acid (Trypsin-EDTA) (200 mg L−1 EDTA, 17,000 U trypsin L−1), penicillin and streptomycin (P/S) (10,000 U mL−1/10 mg mL−1) were purchased from Lonza (Barcelona, ES). Charcoal-dextran stripped fetal bovine serum (FBS-charcoal), Phenol red-free Minimum Essential Medium (MEM) and kanamycin (Kan) were sourced by from PAN-Biotech (Aidenbach, DE). AlamarBlue, 5-carboxyfluorescein diacetate and acetoxy methyl ester (CFDA-AM) were obtained from Life Technologies (Madrid, ES). Sodium dodecyl sulfate (SDS), neutral red (3-amino-7-dimethylamino-2-methylphenanzine hydrochloride) solution (0.33%), 17β-estradiol (E2), glacial acetic acid, in vitro Toxicology Assay Kit Resazurin based, and dimethyl sulfoxide (DMSO) were acquired from Sigma-Aldrich (Madrid, ES). High grade purity water (>18 MΩ/cm) from a Milli-Q Element A10 Century water purification system (Millipore Iberia, ES) was utilized.
3
Isolation and Culture of Human Adipose-Derived Mesenchymal Stem Cells
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Subcutaneous adipose tissue from five healthy human kidney donors became available during the donation procedure. The adipose tissue was collected in minimum essential medium-α (MEM-α) (Sigma-Aldrich, St. Louis, MO) supplemented with 100 IU/ml penicillin, 100 mg/ml streptomycin (P/S) (Lonza, Verviers, Belgium), and 2 mM L-glutamine (Lonza).
The tissue was mechanically disrupted and enzymatically digested with 0.5 mg/ml collagenase type IV in RPMI for 30 min at 37 °C under continuous shaking. Thereafter, the cells were resuspended in MEM-α with 15% fetal bovine serum (FBS; Lonza), 2 mM L-glutamine and 1% P/S, filtered through a 100 µm cell strainer, and transferred to a 175 cm2 culture flasks (Greiner Bio-one, Essen, Germany).
Cultures were kept at 37 °C, 5% CO2, and 95% humidity, at 90% confluence; adherent cells were removed from culture flasks by incubation in 0.05% trypsin-EDTA (Life Technologies, Bleiswijk, The Netherlands) at 37 °C.
Two MSC culture conditions were used for the experiments: unstimulated MSC, and pretreated with IFN-γ (50 ng/ml, Sigma-Aldrich). After incubation for 3 days, MSC were collected to generate cell membrane particles. MSC were used for experiments between passages 2 and 6.
The tissue was mechanically disrupted and enzymatically digested with 0.5 mg/ml collagenase type IV in RPMI for 30 min at 37 °C under continuous shaking. Thereafter, the cells were resuspended in MEM-α with 15% fetal bovine serum (FBS; Lonza), 2 mM L-glutamine and 1% P/S, filtered through a 100 µm cell strainer, and transferred to a 175 cm2 culture flasks (Greiner Bio-one, Essen, Germany).
Cultures were kept at 37 °C, 5% CO2, and 95% humidity, at 90% confluence; adherent cells were removed from culture flasks by incubation in 0.05% trypsin-EDTA (Life Technologies, Bleiswijk, The Netherlands) at 37 °C.
Two MSC culture conditions were used for the experiments: unstimulated MSC, and pretreated with IFN-γ (50 ng/ml, Sigma-Aldrich). After incubation for 3 days, MSC were collected to generate cell membrane particles. MSC were used for experiments between passages 2 and 6.
4
Isolation of Osteoarthritic Chondrocytes
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Human OA articular cartilage was obtained from six patients following total hip arthroplasty (1 male and 5 female patients with a mean ± SD age of 60 ± 13.8 years). Informed consent was obtained from all patients and the study was approved by the Southampton & South West Hampshire Local Research Ethics Committee (LREC 210/01). Full thickness cartilage was dissected from the femoral heads and cut into punches using a 4 mm biopsy punch. Punches were allowed to equilibrate overnight in DMEM/F12 (Life Technologies) supplemented with 5% foetal calf serum (FCS; Invitrogen), 1% insulin–transferrin–selenium (ITS; Sigma–Aldrich), 100 units/mL of penicillin, 100 μg/mL of streptomycin (P/S; Lonza) and 100 μg/mL of ascorbic acid (Sigma–Aldrich), referred to as basal media, in 5% CO2 at 37°C. Only chondrocytes from the superficial layer of OA cartilage were isolated as previously described.29 (link),30 (link)
5
Murine C2C12 Myoblast Expansion Protocol
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Murine C2C12 SkM myoblasts (Blau et al., 1985 (link); Yaffe & Saxel, 1977 (link)) were first seeded (1 × 106 cells) onto pre‐gelatinized (0.2% in dH2O; Type A; Sigma‐Aldrich) T75 flasks (Nunc™; Thermo Fisher Scientific) within a Class II biological safety cabinet (BSC; Kojair) and expanded in growth media composed of high glucose (4.5 g/L) Dulbecco's modified Eagle's medium, including 4 mM l ‐Glutamine (LG; Sigma‐Aldrich), 10% heat‐inactivated fetal bovine serum (hiFBS; SLS), 10% heat‐inactivated newborn calf serum (hiNBCS; Fisher Scientific, Denmark), supplemented with an additional 2 mM LG (Lonza), 100 U/ml penicillin‐100 μg/ml streptomycin (PS; Lonza) in a humidified incubator (HERAcell 150i; Thermo Scientific) at 37°C, 5% CO2 until 80% confluency was attained. Once confluent, cells were washed twice with sterile phosphate‐buffered saline (1× PBS; Sigma‐Aldrich), Trypsinized (0.05% Trypsin/0.02% EDT; Sigma‐Aldrich), and counted using the trypan blue exclusion method (0.4% trypan blue; Sigma‐Aldrich).
6
Differentiation and Stimulation of THP-1 Cells
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The human monocytic leukaemia cell line THP-1 (ECACC) was used routinely between passage 8 and 24 and maintained in complete growth RPMI 1640 medium (Lonza) supplemented with 10% (v/v) FBS (Sera lab), 2 mM L -glutamine, 100 unit/ml penicillin and 100 μg/ml streptomycin (P/S) (Lonza). Cells were kept at 37°C, under 5% CO2 in a humidified incubator.
For THP-1 differentiation into macrophage-like subsets, the cells were plated (at density of 1×106 cell/ml) in complete medium and treated with PMA (M1-like; 25 ng/ml; 3 days followed by 5 days resting in fresh medium) or 1,25-(OH)2-Vitamin D3 (Sigma–Aldrich; M2-like; 10 nM; 7 days) [12 (link)].
Cells were stimulated with 100 ng/ml LPS derived from Escherichia coli K12-LPS (Autogen-Bioclear Ltd.) for the indicated time intervals. Dead cells were aspirated and the live cells and supernatant were harvested by centrifugation at 200g for 5 min, and stored at −20°C until the day of analysis. For cell counting, adherent cells (M1-like) were de-attached by trypsinization using 0.5% (v/v) of trypsin with 0.2% (v/v) EDTA (Sigma–Aldrich).
For THP-1 differentiation into macrophage-like subsets, the cells were plated (at density of 1×106 cell/ml) in complete medium and treated with PMA (M1-like; 25 ng/ml; 3 days followed by 5 days resting in fresh medium) or 1,25-(OH)2-Vitamin D3 (Sigma–Aldrich; M2-like; 10 nM; 7 days) [12 (link)].
Cells were stimulated with 100 ng/ml LPS derived from Escherichia coli K12-LPS (Autogen-Bioclear Ltd.) for the indicated time intervals. Dead cells were aspirated and the live cells and supernatant were harvested by centrifugation at 200
7
Cell Culture of Cancer Cell Lines
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The cancer cell lines AsPC-1 and MIA PaCa-2 were purchased from the American Type Culture Collection (UK). The cancer cell lines A2780 and SK-OV-3 were purchased from the European Collection of Cell Cultures (UK). All cells were grown using distributors' instructions. All cells were cultured in either IMDM, McCoy's 5a Medium Modified or RPMI (Lonza, UK) substituted with 10% FBS (15% for McCoy's 5a Medium Modified) (Bio-Sera, UK) and (v/v); 100 units/mL penicillin, 100 µg/mL streptomycin (P/S) (Lonza, UK). All serum was filtered using a 0.2 µM syringe filter prior to addition to media. When not in use all media was stored between 4-6 °C. All cells were incubated at 37 °C in a 5% CO2 atmosphere. Cells were cultured in tissue culture flasks (Sarstedt, UK) and removed via scraping when cells were 70-90% confluent.
8
Cell Line Cultivation and Maintenance
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The cell lines AsPC-1, Caco-2, Colo320, HCT116, JJN3, Lovo, MCF7, MDA-MB-231, MIA PaCa-2, MM.1S and U266B1 were purchased from the American Type Culture Collection (UK). The cell lines A2780, SK-OV-3, T47D and U937 were purchased from European Collection of Cell Cultures (UK). All cell lines were cultured in accordance with distributor's recommendations. Each cell line was cultured using either DMEM, RPMI, IMDM or McCoy's 5a Medium Modified (Lonza, UK) with FBS (Bio-Sera, UK) and (v/v); 100 units/ml penicillin, 100 µg/ml streptomycin (P/S) (Lonza, UK). All serum, P/S and buffers added to media were filtered through a 0.2µm filter before addition. Between use all media was stored at 2-8°C. All cell lines were incubated at 37°C in a 5% CO2 atmosphere except for MDA-MB-231 which was incubated at 37°C in a 0% CO2 atmosphere. All cell lines were cultured in tissue culture flasks (Sarstedt, UK) and removed when cells were either adherent and in the logarithmic growth phase or removed when at a high enough growth density for suspension cell lines.
9
Gellan Gum Hydrogel Scaffold
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2.1.1 Gellan gum hydrogel scaffolds. GG (low acyl, Mw 1.0 kg/mol; Gelzan CM; Sigma-Aldrich, St. Louis, MO, USA) sterile filtered (0.2 µm) solution of 0.5 % (w/v) concentration was crosslinked with either 16 % (v/v) SPD (BioXtra; Sigma-Aldrich) of 1 mg/mL concentration in 10 % (w/w) sucrose (Sigma-Aldrich) in deionized water or with BaG ext (experimental glass 2-06) [13] (link) containing Dulbecco's Modified Eagle Medium/Ham's Nutrient Mixture F-12 (DMEM/F-12 1:1; Thermo Fisher Scientific, Waltham, MA, USA), 1 % L-glutamine (GlutaMAX; Thermo Fisher Scientific), and 1 % antibiotics/antimycotic containing 100 U/mL penicillin/100 U/mL streptomycin (P/S; Lonza, Basel, Switzerland) to yield GG-BaG hydrogel samples. The cell pellet was resuspended into GG solution at +37°C for hASC encapsulation into 3D hydrogel. The cellhydrogel solution was manually mixed to an ionic crosslinker for immediate gelation. The cell culture medium was added on top of the gelated hASC-laden hydrogel samples.
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