The albino rats were procured from animal house of National Research Centre, Egypt. BM-derived all-nucleated cells were isolated from the femurs of healthy albino rats with an average weight of 120 g according to the method of Lee et al. [24 ]. The isolated cells were expanded by culturing at 10×106 cell density into 100-mm culture dishes (Greiner Bio-One, Austria) in an expansion medium consisting of alpha minimum essential medium (Sigma-Aldrich, USA) containing 20% fetal bovine serum, L-glutamine (2 mM), 2-mercaptoethanol (55 μM), and penicillin/streptomycin. The cells were incubated in 5% CO2 at 37°C for 2-days; the detached cells were discarded by changing the medium and the adherent cells remained in culture for 2-3 weeks. Several passages were performed by subculturing the colony-forming adherent cells in the expansion medium. Subsequently, BM-MSCs were collected at the third passage (P3) on reaching 80% confluence to be exposed for their characterization and proliferation assays.
Alpha minimum essential medium
Manufactured by Merck Group
Sourced in United States
Alpha minimum essential medium is a cell culture medium formulation that provides essential nutrients and components required for the growth and maintenance of various cell types in vitro. It is a widely used tool in cell biology research and applications.
Automatically generated - may contain errors
Lab products found in correlation
Dexamethasone (dex), by Merck Group (4 mentions)
Alpha minimum essential medium, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Sodium ascorbate, by Merck Group (2 mentions)
Glycerophosphate, by Merck Group (2 mentions)
Antibiotic antimycotic, by Merck Group (2 mentions)
Isobutylmethylxanthine, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Insulin, by Merck Group (1 mentions)
Indomethacin, by Merck Group (1 mentions)
β glycerophosphate, by Merck Group (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Hematology analyzer, by Mindray (1 mentions)
Dulbecco s modified eagle s medium dmem, by Merck Group (1 mentions)
Glutamax supplement, by Thermo Fisher Scientific (1 mentions)
Glutamax 1, by Thermo Fisher Scientific (1 mentions)
Ham s f12 medium, by Fujifilm (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (1 mentions)
7 protocols using alpha minimum essential medium
1
Isolation and Expansion of Rat Bone Marrow Mesenchymal Stem Cells
2
Multilineage Differentiation of Wharton's Jelly Mesenchymal Stem Cells
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To confirm the multipotency of WJ-MSCs, osteogenic
and adipogenic differentiation were verified with
alizarin red and oil red O staining respectively. To
induce osteogenesis, WJ-MSCs treated with osteogenic
differentiation medium, alpha minimum essential
medium (Life Technologies, USA) supplemented with
10% FBS (Gibco, USA), 10 mmol/L β-glycerophosphate
(Sigma-Aldrich, USA), 0.1 mmol/L dexamethasone
(Sigma-Aldrich, USA) and 50 mmol/L ascorbic acid
(Sigma-Aldrich, USA) for 21 day. For adipogenesis, the
cells were treated with adipogenic differentiation medium in alpha minimum essential medium supplemented with
10% FBS, 1 mmol/L dexamethasone (Sigma-Aldrich,
USA), 10 mg/mL insulin (Sigma-Aldrich, USA), 0.5
mmol/L isobutyl-methylxanthine (Sigma-Aldrich, USA)
and 100 mmol/L indomethacin (Sigma-Aldrich, USA) for
21 day
and adipogenic differentiation were verified with
alizarin red and oil red O staining respectively. To
induce osteogenesis, WJ-MSCs treated with osteogenic
differentiation medium, alpha minimum essential
medium (Life Technologies, USA) supplemented with
10% FBS (Gibco, USA), 10 mmol/L β-glycerophosphate
(Sigma-Aldrich, USA), 0.1 mmol/L dexamethasone
(Sigma-Aldrich, USA) and 50 mmol/L ascorbic acid
(Sigma-Aldrich, USA) for 21 day. For adipogenesis, the
cells were treated with adipogenic differentiation medium in alpha minimum essential medium supplemented with
10% FBS, 1 mmol/L dexamethasone (Sigma-Aldrich,
USA), 10 mg/mL insulin (Sigma-Aldrich, USA), 0.5
mmol/L isobutyl-methylxanthine (Sigma-Aldrich, USA)
and 100 mmol/L indomethacin (Sigma-Aldrich, USA) for
21 day
3
Quantifying Mesenchymal Stem Cell Enrichment
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Before and after filtration in the SECCS, 10-mL samples of bone marrow were collected for culture for each sample. The samples were suspended in 2.5 mL alpha-minimum essential medium (Sigma, Santa Clara, California, USA) containing 10% fetal bovine serum (Hyclone, Logan, Utah, USA), 50 mg/mL sodium ascorbate (Sigma), 1% antibiotic/antimycotic, 10 mM glycerophosphate (Sigma), and 10−8 M dexamethasone (Sigma) and cultured in 6-well plates (0.2 mL per well, replicated in 3 wells) at 37 °C in a humidified atmosphere with 5% CO2. Medium was refreshed every 2 days. On day 10, alkaline phosphatase (ALP) staining was performed to stain the colony-forming units (CFUs) expressing ALP activity (ALP + CFU). The number of ALP + CFU indicates the number of MSCs in pre- or post-SECCS bone marrow18 (link),37 (link). ALP + CFU (diameter, ≥2 mm) numbers were counted by 2 independent experienced researchers. The ALP + CFU number was the mean of 3 wells. The difference between ALP + CFU numbers of pre- and post-SECCS bone marrow was determined for each patient. The formula to count the MSC screening-enrichment rate is (PRE ALP+ CFU − POST ALP+ CFU)/PRE ALP+ CFU × 100%.
4
Quantifying Mesenchymal Stem Cell Enrichment
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Ten milliliters of pre- and post- enrichment bone marrow was collected for MSC adherent culture as reported by other researchers16 (link),17 (link). In brief, samples were cultured in six-well plates with osteogenic induction solution [alpha-minimum essential medium (Sigma, Santa Clara, California, USA) suspended in 10% fetal bovine serum (Hyclone, Logan, Utah, USA), 50 mg/mL sodium ascorbate (Sigma), 1% antibiotic/antimycotic (Sigma), 10 mM glycerophosphate (Sigma), and 10-8 M dexamethasone (Sigma)]. The medium was changed every 2 days, and at 10–14 days, the cultures were washed by phosphate-buffered saline (PBS), fixed by 4% paraformaldehyde and then stained with an alkaline phosphatase (ALP) staining kit (Lexiang, Shanghai, China), which as reported before was used for calculating colony-forming units (CFUs). Each CFU that was 2 mm in diameter or larger was manually quantified as one count. The mean count of ALP-positive colonies from six individual wells was used for each sample, and the presence of ALP+CFU represented an MSC colony18 (link). Enrichment efficiency was formulated as (PREALP+CFUs – POSTALP+CFUs)/ PREALP+CFUs×100%. Another 1 mL bone marrow pre- and post-enrichment was sent for routine blood examination by a hematology analyzer (Mindray, China) to evaluate the adhesion of other cells in bone marrow, including leukocytes, erythrocytes, and platelets.
5
Diverse Cell Culture Conditions
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All media for cell cultures were supplemented with 10% fetal calf serum (FCS) unless otherwise specified. All cell lines were grown at 37°C under 5% CO2 in air. The MC3T3-E1 (mouse osteoblast) and T98G (human glioblastoma) cell lines, provided by K. Irie, Fukuoka University, were cultured in alpha minimum essential medium (Gibco Laboratories). The Balb3T3 (clone A31) (mouse embryo fibroblast), Vero (African green monkey kidney epithelial), COS7 (African green monkey kidney fibroblast), and Rat-1 (rat fibroblast) cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich). P19 (mouse embryonal carcinoma) cells were cultured in alpha minimum essential medium (Sigma-Aldrich) containing 1% GlutaMAX supplement (catalog number 35050-061; Gibco Laboratories). Differentiation of P19 cells was stimulated by incubation in the presence of retinoic acid (RA) at 500 nM as described previously (28 (link)). NTera2/cl.D1 (NT2) (human embryonal carcinoma) cells were cultured in DMEM plus GlutaMAX-I (catalog number 10566-016; Gibco Laboratories). CHO-K1 (Chinese hamster ovary epithelial) cells were grown in Ham’s F-12 medium (Wako). 293FT (human embryo kidney epithelial) and Plat-E (human embryo kidney epithelial) cells were cultured according to the manufacturer’s instructions.
6
Differentiation of mDP E6 Cells into Odontoblasts
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The mDP E6 mouse dentate papilla cell line was used for analysis of differentiation into odontoblasts as described previously [13] . Briefly, mDP E6 cells were cultured in alpha minimum essential medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (Sigma-Aldrich), 100 units/mL penicillin (Thermo Fisher), and 100 mg/mL streptomycin (Thermo Fisher) in a humidified atmosphere of 5% CO 2 at 37 C. Differentiation into odontoblasts was induced by culturing for five days in the presence of 10 nM dexamethasone (Sigma-Aldrich), 1.8 mM potassium hydrogen phosphate (Nakarai Tesque), 100 mM ascorbic acid, and 10 ng/mL basic fibroblast growth factor (Peprotech, Rocky Hill, NJ, USA).
7
Coral Scaffold for Bone Regeneration
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Two hundred to 300 μm calcium carbonate particles (Coral from Porites sp.; Biocoral®, Inoteb, France) were used as the bioceramic scaffolds. These particles were sterilized by autoclaving at 120°C for 20 min. hMSC isolation and culture HMSCs were harvested from bone marrow obtained as discarded tissue during routine bone surgery from three patients (one woman and two men, 15, 22, and 31 years of age, respectively) at the Lariboisiere Hospital (Paris, France). The tissues were collected with the respective donor's consent in agreement with Lariboisiere Hospital regulations. hMSCs were isolated from each donor bone marrow using a procedure adapted from literature reports20 and were characterized for the expression of select CD markers (specifically, positive for CD90, CD73, CD105, and negative for CD45; data not shown). These cells were cultured in Alpha-Minimum Essential Medium (Sigma) containing 10% fetal bovine serum (PAA Laboratories) and antibiotics (PAA Laboratories) into tissue culture plates. Adherent cells from each donor were cultured separately until passage 3 and then pooled before seeding on coral scaffolds.
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