MSC lines were generated from five subjects. Bone marrow MNCs were obtained by Ficoll-1077 density gradient separation. BM-MNCs were seeded at a density of 8 to 23 × 104 cells cm−2 in α-MEM medium (Sigma-Aldrich, St Louis, MO, USA) supplemented with heparin (3.5 IU ml−1) and 5% platelet lysate. Platelet lysate was prepared by freeze-thawing of platelets (>0.8 × 109 platelets per ml), followed by centrifugation at 4,700 g and collection of the supernatant. At 7 days after seeding, the culture medium was refreshed. Subsequently, cells were passed when 80% confluency was reached. After 7 days of culture, all floating and dead cells were washed away and a layer with MSCs remained. MSCs were cultured for up to 5 passages.
α mem medium
Manufactured by Merck Group
Sourced in United States, United Kingdom
α-MEM medium is a cell culture medium commonly used in laboratory settings. It provides the necessary nutrients and components to support the growth and maintenance of various cell types in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Merck Group (3 mentions)
Streptomycin, by Merck Group (3 mentions)
Penicillin, by Merck Group (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Dexamethasone (dex), by Merck Group (3 mentions)
Murine m csf, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Merck Group (2 mentions)
Dentin discs, by Immunodiagnostic Systems (2 mentions)
Evos xl inverted microscope, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by American Type Culture Collection (2 mentions)
Dmi6000b inverted microscope, by Leica (1 mentions)
Recombinant murine m csf, by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Rankl, by Merck Group (1 mentions)
Murine rankl, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Trypsin edta, by Merck Group (1 mentions)
α mem, by Merck Group (1 mentions)
M csf, by Sino Biological (1 mentions)
28 protocols using α mem medium
1
Isolation and Expansion of Mesenchymal Stem Cells
2
In vitro Osteoclast Differentiation and Resorption Assay
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In vitro osteoclast cultures were performed essentially as described before (54 (link), 55 (link)). Bone marrow cells obtained by flushing the tibia and femur of wild type or mutant mice were cultured in the presence of 10 ng/ml murine M-CSF (Peprotech) for 2 days in α-MEM medium (Sigma) supplemented with 10% FCS (Gibco) and antibiotics. Non-adherent cells were then plated at the concentration of 1.5 × 105 cells/cm2 and cultured in the presence of 50 ng/ml recombinant murine M-CSF and 50 ng/ml murine RANKL (Peprotech) with medium changes every 2 days. In parallel macrophage cultures, the cells were cultured under identical conditions except that RANKL was omitted.
Cultures were terminated and osteoclast-specific staining was performed using a commercial tartrate-resistant acid phosphatase (TRAP) staining kit (Sigma-Aldrich) at the indicated times after the first addition of RANKL. Photomicrographs were taken using a Leica DMI6000B inverted microscope. The images were then analyzed either manually or by the ImageJ software. Osteoclasts were defined as TRAP-positive cells with 3 or more nuclei.
For in vitro resorption assays, osteoclasts were cultured under similar conditions for 7 days on an artificial hydroxyapatite surface (Sigma-Aldrich) followed by washing, imaging by dark field microscopy and further analysis by ImageJ software.
Cultures were terminated and osteoclast-specific staining was performed using a commercial tartrate-resistant acid phosphatase (TRAP) staining kit (Sigma-Aldrich) at the indicated times after the first addition of RANKL. Photomicrographs were taken using a Leica DMI6000B inverted microscope. The images were then analyzed either manually or by the ImageJ software. Osteoclasts were defined as TRAP-positive cells with 3 or more nuclei.
For in vitro resorption assays, osteoclasts were cultured under similar conditions for 7 days on an artificial hydroxyapatite surface (Sigma-Aldrich) followed by washing, imaging by dark field microscopy and further analysis by ImageJ software.
3
Murine Bone Marrow-Derived Macrophage Isolation
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The tibiae of male mice (6–7 weeks old) were aseptically collected, and the marrow were flushed out with basic medium (1640 medium containing 10% fetal bovine serum (ExCellent Bio, Australia) and 1% v/v penicillin/streptomycin (HyClone, USA)). The marrow suspension was subsequently sieved with a 40 µm cell strainer and treated with lysate (Beyotime, China), and subjected to culture with the basic medium with the presence of 70 ng mL−1 M‐CSF (Sino Biological, China). Non‐adherent cells were collected 24 h later and cultured for 3 days to get the adherent cells ready as mBMDMs. The cells were harvested with a cell scraper for further use.
CRL‐12424 cells (a mesenchymal stem cell line derived from mouse bone marrow) were purchased from ATCC and maintained in basic α‐MEM medium consisting of α‐modified eagle medium (α‐MEM, SIGMA, UK) supplemented with 10% fetal bovine serum and 1% v/v penicillin/streptomycin. The cells were harvested with 0.25% trypsin/EDTA (SIGMA, UK) for further use.
CRL‐12424 cells (a mesenchymal stem cell line derived from mouse bone marrow) were purchased from ATCC and maintained in basic α‐MEM medium consisting of α‐modified eagle medium (α‐MEM, SIGMA, UK) supplemented with 10% fetal bovine serum and 1% v/v penicillin/streptomycin. The cells were harvested with 0.25% trypsin/EDTA (SIGMA, UK) for further use.
4
Isolation and Characterization of Decidua Mesenchymal Stromal Cells
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DMSC were isolated from the decidua basalis that remained attached to the placenta using previously published methods18 (link)20 (link). DMSC were cultured in α-MEM medium (Sigma-Aldrich) with 10% FBS, 100 U/ml penicillin, 100 mg/ml streptomycin and 2 mM L-Glutamine (Sigma Aldrich).
DMSC23 cell lines were created by human telomerase reverse transcriptase (hTERT) transduction of the primary DMSC40 (link). DMSC23 cells were maintained in culture using Mesencult Proliferation Kit (Stem Cell Technologies) which consists of Mesencult basal medium, 10% Mesencult supplement, 100 U/ml penicillin, and 100 mg/ml streptomycin. All cell cultures were maintained at 37 °C with 5% CO2 in a humidified incubator. DMSC and DMSC23 cells were characterised by flow cytometry for expression of CD90, CD146, CD166, CD44, CD73, CD105 and were negative for CD45, CD19, and HLA-DR. The cells displayed multi-lineage differentiation potential along the osteogenic, adipogenic, and chondrogenic lineages as shown previously18 (link)19 40 (link). Primary cell and cell lines were passaged after reaching 80% confluency. At each passage, cells were harvested using TrypLE Express solution (Life Technologies) and cultured on uncoated tissue culture flasks. All cultured primary cells were used for experiments up to P5 and DMSC23 cell lines up to P30.
DMSC23 cell lines were created by human telomerase reverse transcriptase (hTERT) transduction of the primary DMSC40 (link). DMSC23 cells were maintained in culture using Mesencult Proliferation Kit (Stem Cell Technologies) which consists of Mesencult basal medium, 10% Mesencult supplement, 100 U/ml penicillin, and 100 mg/ml streptomycin. All cell cultures were maintained at 37 °C with 5% CO2 in a humidified incubator. DMSC and DMSC23 cells were characterised by flow cytometry for expression of CD90, CD146, CD166, CD44, CD73, CD105 and were negative for CD45, CD19, and HLA-DR. The cells displayed multi-lineage differentiation potential along the osteogenic, adipogenic, and chondrogenic lineages as shown previously18 (link)19 40 (link). Primary cell and cell lines were passaged after reaching 80% confluency. At each passage, cells were harvested using TrypLE Express solution (Life Technologies) and cultured on uncoated tissue culture flasks. All cultured primary cells were used for experiments up to P5 and DMSC23 cell lines up to P30.
5
Melatonin and Luzindole Effects on Mouse Embryo Development
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Female mice were superovulated by an intra-peritoneal (IP) injection of 7.5 IU pregnant mare serum gonadotropin (PMSG, G 4877, Sigma-Aldrich, USA), followed by injection of 7.5 IU human chorionic gonadotropin (HCG, Karma, Germany) 48 hr later (10 (link)).
To ensure the reliability of gestation time, female mice were paired overnight with males of proven fertility (ratio = 1:1) and females with a vaginal plug were selected for the study.
At 42-48 hr after the HCG injection, the embryos were flushed into Ham’s F10 (Sigma, USA) medium under a stereomicroscope (Nikon SMZ-2T, Japan) (11 (link)).
After three washes, embryos with normal develop-mental morphologies were randomly divided into 3 groups including:
To ensure the reliability of gestation time, female mice were paired overnight with males of proven fertility (ratio = 1:1) and females with a vaginal plug were selected for the study.
At 42-48 hr after the HCG injection, the embryos were flushed into Ham’s F10 (Sigma, USA) medium under a stereomicroscope (Nikon SMZ-2T, Japan) (11 (link)).
After three washes, embryos with normal develop-mental morphologies were randomly divided into 3 groups including:
α-MEM medium (Sigma, USA) containing 10% fetal bovine serum (FBS) and 10-9 M melatonin as the melatonin-treated group.
α-MEM medium with serum and without melatonin as the simple media group.
α-MEM medium containing serum and 10-9 M luzindole as the Luzindole-treated group.
6
Isolation and characterization of rat MSCs
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Mesenchymal stem cells were obtained from the bone marrow of adult Lewis 1A rats as previously described [12 (link),16 (link),17 (link)]. The ends of femurs and tibias were cut, and bone marrow cells (BMC) were aspirated with a 25-gauge needle. To remove all the BMC, the bone canal was flushed with αMEM medium (Sigma-Aldrich, Saint-Quentin Fallavier, France) supplemented with 20% foetal calf serum (FCS; Gibco, Life technologiies ltd, Asint-Aubin, France), 100 U/ml penicillin (Sigma-Aldrich) and 100 μg/ml streptomycin (Sigma-Aldrich). MSC were selected by an overnight incubation of the BMC onto uncoated plastic dishes. Plastic-adherent cells were then plated at a density of 8 × 103 cells/cm2 and split at 80% confluency. Full characterization of the cells was previously described [12 (link),17 (link)] and current analyses by flow cytometry indicate that over 95% of the cells were consistently positive for CD90 and negative for CD45. MSC were used at passage 4.
7
Scoring of Oocyte Nucleolus Heterochromatin
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GV oocytes were scored for the presence/ absence of a Hoechst-positive ring of heterochromatin surrounding the nucleolus, leading to allocation in two classes (Surrounded Nucleolus, SN; Not Surrounded Nucleolus, NSN). Scoring was performed while holding the oocytes in Hepes-buffered CZB medium in the presence of 0.1 μg/mL Hoechst 33342 on the stage of an inverted microscope (Nikon TE 2000) fitted with an EXFO light source. After sorting, oocytes were allowed to progress to MII by culturing them in α-MEM medium (Sigma cat.no. M4526) supplemented with 0.2% BSA (w/v) and 50 mg/ mL gentamicin. Incubation took place at 37°C under 5% CO2 and 5% O2 (balance N2). This low-oxygen atmosphere was achieved in a homemade plastic chamber positioned on the shelf of the incubator and connected to a 5/5/90 CO2/O2/N2 tank.
8
Lycopene and Curcumin Bioactivity Assay
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Crystalline lycopene preparations, purified from tomato extract (>97%), were supplied by Lycored Ltd. (Beer Sheva, Israel). Tetrahydrofuran (THF), containing 0.025% butylated hydroxytoluene (BHT) as an antioxidant, was purchased from Aldrich (Milwaukee, WI, USA). fetal calf serum (FCS), sodium pyruvate, and Ca2+/Mg2+-free PBS were purchased from Biological Industries (Beth Haemek, Israel). DMEM medium was purchased from Gibco (Grand Island, NY, USA). α-MEM medium, Dimethyl sulfoxide (DMSO), P-nitrophenyl phosphate and acid phosphatase leukocyte kit (387A) were purchased from Sigma Chemicals. Curcumin was purchased from Cayman Chemicals (Ann Arbor, MI, USA). Carnosic acid was purchased from Alexis Biochemicals (Läufenfingen, Switzerland).
9
Isolation of Decidua Mesenchymal Stem Cells
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We have previously reported the isolation of DMSCs from the decidua basalis adhering onto the maternal side of the placenta [13 ]. About eight grams of placental tissue was dissected from the basal plate, washed four times in PBS, finely minced and digested in trypsin (0.25%; Life Technologies, CA, USA) and DNAse 1 (50 μg/mL; Worthington, NJ, USA) at 4°C overnight. Fetal bovine serum (FBS; Thermo Scientific, MA, USA) was added to inactivate the trypsin and the digest was centrifuged at 200g for 5 min. The pelleted tissue was digested in type 1 collagenase (10 mg/mL; Worthington) and DNAse 1 (50 μg/mL, Worthington) for 30 min at 37°C and strained through a 100 μm stainless steel sieve. The filtrate was layered over Histopaque (Sigma-Aldrich, MO, USA) and separated by density gradient centrifugation at 400g for 30 min. Mononuclear cell layers containing the DMSCs were aspirated and centrifuged at 200g for 5 min. DMSCs were maintained in α-MEM medium (Sigma-Aldrich) with 10% FBS, penicillin/streptomycin (100 U/mL and 100 mg/mL, respectively; Life Technologies) and 2 mM L-glutamine (Sigma-Aldrich). P0 DMSCs were passaged after reaching 80% confluence and cells were expanded up to P5.
10
Isolation of Murine Peritoneal Macrophages
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Macrophages were obtained from the peritoneal cavity of mice. To obtain resident macrophages under resting conditions, we injected 5 ml of sterile ice-cold PBS (pH 7.4) into the peritoneal cavity and aspirated the fluid to harvest peritoneal cells. The cells were washed twice with α-MEM medium (Sigma) containing 10% FBS. After 1 hour of culture, nonadherent cells were removed, and after 24 hours of culture, adherent cells were harvested and used as macrophages.
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