REF cells were maintained in alpha-modified Eagle medium (Gibco BRL) supplemented with 5% (v/v) fetal bovine serum (FBS), penicillin (10,000 units/ml), and streptomycin (1 mg/ml). HeLa and A375 cells were maintained in Dulbecco’s-modified Eagle medium supplemented with 10% FBS. Transient transfection was carried out using Effectene (Qiagen, Hilden, Germany) or Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.
Alpha modified eagle medium
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alpha-modified Eagle medium is a cell culture medium formulation designed to support the growth and maintenance of various cell types in vitro. It provides a balanced combination of nutrients, amino acids, vitamins, and other essential components required for cell proliferation and survival.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Tissue culture flasks, by Sarstedt (2 mentions)
Ascorbic acid 2 phosphate, by Merck Group (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Effectene, by Qiagen (1 mentions)
Dispase, by Thermo Fisher Scientific (1 mentions)
Pth 1 34, by Merck Group (1 mentions)
Collagenase type 1, by Thermo Fisher Scientific (1 mentions)
L ascorbic acid, by Merck Group (1 mentions)
Human bone marrow derived mesenchymal stem cells, by Lonza (1 mentions)
Nanoscope 5 dimension 3100 microscope, by Veeco (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin and streptomycin, by Merck Group (1 mentions)
Hbmsc, by Lonza (1 mentions)
L ascorbic acid 2 phosphate, by Fujifilm (1 mentions)
Raw264.7 cells, by Thermo Fisher Scientific (1 mentions)
Raw264.7 cell, by American Type Culture Collection (1 mentions)
8 protocols using alpha modified eagle medium
1
Cell Culture and Transfection Protocols
2
Isolation and Characterization of Rat BMSCs
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BMSCs were obtained from donor rats as described (Shen et al., 2006 (link); Guo et al., 2011 (link)). The rats were euthanized with CO2 and both ends of the tibiae, femurs and humerus were cut off by scissors. A syringe fitted with an 18-gauge needle was inserted into the shaft of the bone and bone marrow was flushed out with culture medium (alpha-modified Eagle medium, Gibco, Carlsbad, CA, USA; 10% fetal bovine serum, Hyclone, Logan, UT, USA). The bone marrow was then mechanically dissociated and the suspension passed through a 100-μm cell strainer to remove debris. The cells were incubated at 37°C in 5% CO2 in tissue-culture flasks (100 × 200 mm; Sarstedt, Nümbrecht, Germany), and non-adherent cells removed by replacing the medium. At day 7, when the cultures reached 80% confluence, the cells were washed with PBS and harvested. The cell numbers were calculated by the Hemocytometer. For intravenous administration, 1.5 × 106 cells (1.5 M) in 0.2 ml PBS were slowly injected into one tail vein of the anesthetized rat over a 2-min period using a 22-gauge needle. The property of expanded cells was assessed by flow cytometry with conventional markers (Guo et al., 2011 (link)). Flow cytometry analyses were performed at the University of Maryland GreenBaum Cancer Center Shared Flow Cytometry Facility.
3
Isolation and Systemic Delivery of Rat Bone Marrow Stromal Cells
4
Isolation and Culture of PDLSCs
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Healthy premolars were collected from 10 adults (15–20 years of age; five men and five women) for orthodontic purposes at the Department of Oral and Maxillofacial Surgery, the Affiliated Stomatological Hospital of Sun Yat-sen University. All participants provided informed consent for the collection and use of their tissues. The protocols were approved by the University Ethics Committee.
PDLSCs were isolated and cultured as previously reported [6 (link)]. Briefly, periodontal tissue was gently separated from the surface of the middle third of the root and then digested with 3 mg/mL collagenase type I and 4 mg/mL dispase (Gibco-BRL, Gaithersburg, MD, USA) at 37°C for 1 h. Colony-forming cells were collected and then cultured in alpha modified Eagle medium (Gibco-BRL) supplemented with 10% fetal bovine serum (Gibco-BRL), 100 μg/mL streptomycin, 100 U/mL penicillin (Hyclone, Logan, UT, USA), 200 μM l -ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA), and 5 mM l -glutamine (Gibco-BRL) at 37°C in an atmosphere containing 5% CO2. The medium was changed every 3 days. Cells were used at passages 3–5. For intermittent PTH treatment, cells were treated with 10−12 M PTH (1-34) (Sigma-Aldrich) for 6 h, followed by another 6 h treatment after 32 h. Cells were harvested at various times after treatment with 10−12 M PTH (1-34) for RNA and protein isolation.
PDLSCs were isolated and cultured as previously reported [6 (link)]. Briefly, periodontal tissue was gently separated from the surface of the middle third of the root and then digested with 3 mg/mL collagenase type I and 4 mg/mL dispase (Gibco-BRL, Gaithersburg, MD, USA) at 37°C for 1 h. Colony-forming cells were collected and then cultured in alpha modified Eagle medium (Gibco-BRL) supplemented with 10% fetal bovine serum (Gibco-BRL), 100 μg/mL streptomycin, 100 U/mL penicillin (Hyclone, Logan, UT, USA), 200 μM
5
Human Mesenchymal Stem Cell Culture
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Human bone marrow-derived
mesenchymal stem cells (Lonza) were used for the cell experiments.
The growth medium consisted of Alpha modified Eagle medium (Gibco),
10% (v/v) fetal bovine serum (Gibco), and 0.1% (v/v) ascorbic acid
2-phosphate (Sigma). Cells were incubated at 37 °C, 5% CO2. The cells were harvested at approximately 80–90%
confluency from T75 culture flasks by trypsin for 3–5 min at
37 °C for further subcultures.
mesenchymal stem cells (Lonza) were used for the cell experiments.
The growth medium consisted of Alpha modified Eagle medium (Gibco),
10% (v/v) fetal bovine serum (Gibco), and 0.1% (v/v) ascorbic acid
2-phosphate (Sigma). Cells were incubated at 37 °C, 5% CO2. The cells were harvested at approximately 80–90%
confluency from T75 culture flasks by trypsin for 3–5 min at
37 °C for further subcultures.
6
Topographic Characterization of MSC Culture
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Topography features were characterized by atomic force microscope (Nanoscope V Dimension 3100 microscope, Veeco, United States) operating in tapping mode in air (model DNP-10 tip). To determine the wavelength and amplitude of the topographies, the obtained AFM images were analyzed using NanoScope Analysis software.
Cell culture hBM-MSCs ( passage 2) were obtained from Lonza and cultured in growth medium containing Alpha modified Eagle medium (Gibco), 10% (v/v) fetal bovine serum (Gibco), 0.1% ascorbic acid 2-phosphate (Sigma) and 1% penicillin/streptomycin (Gibco). Cells were incubated in T75 culture flasks at 37 °C in a humidified atmosphere with 5% CO 2 . Culture medium was changed every 3 days and cells were harvested at ≈80% confluency. The confluent cells were routinely subcultured by trypsinization. MSCs of passage 4 were used for seeding onto the topographies and the differentiation experiments.
Cell culture hBM-MSCs ( passage 2) were obtained from Lonza and cultured in growth medium containing Alpha modified Eagle medium (Gibco), 10% (v/v) fetal bovine serum (Gibco), 0.1% ascorbic acid 2-phosphate (Sigma) and 1% penicillin/streptomycin (Gibco). Cells were incubated in T75 culture flasks at 37 °C in a humidified atmosphere with 5% CO 2 . Culture medium was changed every 3 days and cells were harvested at ≈80% confluency. The confluent cells were routinely subcultured by trypsinization. MSCs of passage 4 were used for seeding onto the topographies and the differentiation experiments.
7
Human Bone Marrow Stem Cell pH Modulation
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hBMSCs were purchased from Lonza (Walkersville, MD, USA) and cultured in alpha-Modified Eagle Medium (Invitrogen, Carlsbad, CA, USA) containing 15% fetal bovine serum (FBS, Invitrogen), 100 mM L-ascorbic acid 2-phosphate (Wako Pure Chemical Industries, Osaka, Japan), 1% penicillin and streptomycin (Sigma), and 1% L-glutamine (Invitrogen). Media were titrated to pH 7.4, pH 6.8, or pH 6.4 by adding 20 mM HEPES and 1 M HCl, as described previously [10 (link),16 (link)]. Media were retitrated after incubation for 24 h, before using in the experiments. Cells were then cultured under different pH conditions for 48 h, before analysis of the stem cell characteristics and functions.
8
Osteoclast Differentiation from RAW 264.7 Cells
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