Human placenta stem cells (hPSCs) were obtained from CHA Biotech (Seongnam, Republic of Korea). Human PSCs were maintained with minimum essential medium (MEM)-alpha GlutaMAX (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS (Thermo Fisher Scientific), 1% penicillin/streptomycin (Thermo Fisher Scientific), 25 ng/mL human fibroblast growth factor 4 (Peprotech Inc., Rocky Hill, NJ, USA), and 1 μg/mL heparin (Sigma-Aldrich, St. Louis, MO, USA). To improve the recovery effect of hPSCs, hPSCs were incubated in 2.2% O2 and 5.5% CO2 conditions for 30 min. After exposure, the hPSCs and hHPPSCs were incubated in MEM-alpha GlutaMAX containing 10% exosome-free FBS (ThermoFisher Scientific) with supplements for 48 h. We isolated extracellular vesicles (EVs) from hHPPSCs (passages of 8–10) using ExoQuick-TC kit (SYSTEM BIOSCIENCES (SBI), Palo Alto, CA, USA). The experimental process followed the manual provided by the company. The exosome obtained in the final step was dissolved in 100 μL PBS and quantified by the BCA method. The EVs were stored at −80 ℃. Using PMX-120 ZetaView® Mono Laser (Particle Metrix, Meerbusch, North Rhine-Westphalia, Germany), the size of EVs was measured.
Alpha mem glutamax
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
Alpha-MEM GlutaMax is a cell culture medium used for the growth and maintenance of various cell types. It is formulated to provide essential nutrients, amino acids, and other components necessary for optimal cell growth and proliferation.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (12 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (11 mentions)
Trypsin edta, by Thermo Fisher Scientific (4 mentions)
Ciprofloxacin hcl, by Bioworld Technology (3 mentions)
Heparin, by Merck Group (2 mentions)
Human fibroblast growth factor 4, by Thermo Fisher Scientific (2 mentions)
Fetal calf serum (fcs), by Merck Group (2 mentions)
Collagenase 4, by Merck Group (1 mentions)
Exoquick tc kit, by System Biosciences (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Microtome blade, by Leica Biosystems (1 mentions)
Techno glass, by AGC Techno Glass (1 mentions)
30 mm cell culture insert, by Merck Group (1 mentions)
Trypsin edta 1x, by Thermo Fisher Scientific (1 mentions)
Glutamax medium, by Thermo Fisher Scientific (1 mentions)
Tazemetostat, by Selleck Chemicals (1 mentions)
Antibiotic antimycotic anti anti, by Thermo Fisher Scientific (1 mentions)
Normocin, by InvivoGen (1 mentions)
Chang medium d, by Irvine Scientific (1 mentions)
21 protocols using alpha mem glutamax
1
Isolation and Characterization of Exosomes from Human Placenta Stem Cells
2
Osteoclast Differentiation from PBMCs
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Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation (density: 1.077 g/mL; PanBiotech, Aidenbach, Germany) and washed twice with DPBS (Gibco).
Cells were then resuspended in alphaMEM Glutamax (Gibco, Life technologies, Darmstadt, Germany) supplemented with 10% FCS, 100 U/mL penicillin (Life technologies, Darmstadt, Germany), 10 μg/mL streptomycin (Life technologies), and 25 ng/mL MCSF (BioLegend, Amsterdam The Netherlands). The cells were seeded at specific concentrations, depending on the culture plate used (1.5 × 106 cells/well in 6 well plates, 0.15 × 106 cells/well in 48 well plates, and 0.05 × 106 cells/well in 96 well plates) and cultured at 37 °C, 5% CO2. After 24 h, the medium was replaced with a differentiation medium containing alphaMEM Glutamax (Gibco, Life technologies, Darmstadt, Germany) supplemented with 10% FCS, 100 U/mL penicillin (Life technologies), 10 μg/mL streptomycin (Life technologies, Darmstadt, Germany), 25 ng/mL MCSF, and 50 ng/mL RANKL (BioLegend, Amsterdam The Netherlands). As an osteoclast differentiation control, cells were cultured without RANKL. The cells were cultured for 14 days, and the medium was replaced twice a week.
Cells were then resuspended in alphaMEM Glutamax (Gibco, Life technologies, Darmstadt, Germany) supplemented with 10% FCS, 100 U/mL penicillin (Life technologies, Darmstadt, Germany), 10 μg/mL streptomycin (Life technologies), and 25 ng/mL MCSF (BioLegend, Amsterdam The Netherlands). The cells were seeded at specific concentrations, depending on the culture plate used (1.5 × 106 cells/well in 6 well plates, 0.15 × 106 cells/well in 48 well plates, and 0.05 × 106 cells/well in 96 well plates) and cultured at 37 °C, 5% CO2. After 24 h, the medium was replaced with a differentiation medium containing alphaMEM Glutamax (Gibco, Life technologies, Darmstadt, Germany) supplemented with 10% FCS, 100 U/mL penicillin (Life technologies), 10 μg/mL streptomycin (Life technologies, Darmstadt, Germany), 25 ng/mL MCSF, and 50 ng/mL RANKL (BioLegend, Amsterdam The Netherlands). As an osteoclast differentiation control, cells were cultured without RANKL. The cells were cultured for 14 days, and the medium was replaced twice a week.
3
Isolation and Culture of Human Placental Mesenchymal Stem Cells
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We previously described the methods for preparing hPMSCs [22 (link)]. The collection and use of the samples were approved by the Institutional Review Board of CHA General Hospital (Seoul, South Korea). All of the participants provided written informed consent prior to the sample collection. Briefly, the placentas were collected from mothers who were free of medical, obstetric, and surgical complications who delivered at term (> 37 gestational weeks). The hPMSCs were collected from the inner side of the chorioamniotic membrane of the placenta and then treated with 0.5% collagenase IV (Sigma-Aldrich, St. Louis, MO, USA). Human PMSCs were cultured in Minimum Essential Media (MEM) alpha GlutaMAX (Thermo Fisher Scientific) supplemented with 10% FBS (Thermo Fisher Scientific), 1% penicillin/streptomycin (Thermo Fisher Scientific), 25 ng/mL human fibroblast growth factor 4 (Peprotech, Inc., Rocky Hill, NJ, USA), and 1 μg/mL heparin (Sigma-Aldrich).
4
Cell Culture Protocols for Melanoma and Prostate Cancer
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In this study, human primary melanoma (MM418-C1) cells (QIMR, Brisbane, Australia) and DU145 prostate cancer cells (human epithelial cells ATCC® HTB-81™) were used. MM418-C1 were cultured and maintained in RPMI 1640 (Gibco®), 10% FBS, and 1% penicillin-streptomycin. DU145 cells were cultured and maintained in MEM Alpha + GlutaMAX™ and 15-millimolar HEPES (Thermo Fisher Scientific, Waltham, MA, USA) and 10% FBS (Gibco®), and 1% antibiotics (penicillin-streptomycin; Gibco®). The cell lines were initially cultured and grown to about 80% confluence in a 75 cm3 flask, and then were subcultured in a 1:3 ratio with trypsin EDTA (Gibco®). The incubation conditions during the experiments were 37 °C with 5% CO2 in a humidified environment.
5
Culturing A549 and Du145 Cancer Cells
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In this research, A549 lung cancer cells (human epithelia cells ATCC® CCL-185™) and Du145 prostate cancer cells (human epithelial cells ATCC® HTB-81™) were used. A549 cells were cultured and maintained in DMEM/F12 supplemented with L-glutamine and 15 mM HEPES (Thermo Fisher Scientific, Waltham, MA, USA) and 10% fetal bovine serum (FBS) (Thermo Fisher Scientific) and 1% antibiotics (Penicillin-Streptomycin) (Thermo Fisher Scientific). Du145 cells were cultured and maintained in MEM Alpha + GlutaMAX™ and 15 mM HEPES (Thermo Fisher Scientific) and 10% FBS (Thermo Fisher Scientific) and 1% antibiotics (Penicillin-Streptomycin; Thermo Fisher Scientific). Both the cell lines were initially cultured and grown to about 80% confluency in a 75 cm2 flask and then were subcultured in 1:3 ratio by trypsin EDTA (Thermo Fisher Scientific). Incubation condition during the experiments was 37°C with 5% CO2 in a humidified environment.
6
Ex Vivo Ovarian Tissue Slicing and Culture
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Ovaries from 4-week-old mice were sliced at a thickness of several hundred micrometers using a microtome blade (Leica Biosystems, Nussloch, Germany) under a stereoscopic microscope (Leica Biosystems). Ovarian tissue slices were
placed in a 30-mm cell culture insert (Merck Millipore, Darmstadt, Germany), which was subsequently placed in a 3.5-cm culture dish (AGC Techno Glass, Shizuoka, Japan). Ovarian slices were cultured in the minimum essential medium
alpha (MEM-alpha) GlutaMax (Gibco, Carlsbad, CA, USA) supplemented with 5% (v/v) fetal bovine serum (FBS, Gibco), 100 mIU/ml FSH from human pituitary gland (Sigma-Aldrich, St Louis, MO, USA), and 10 mIU/ml LH from equine pituitary
gland (Sigma-Aldrich) under conditions of 5% CO2 and 37°C. The cultured ovarian tissue slices were treated with 100 mIU/ml of LH for 12 h every 4 days to reproduce the physiological LH surge. Concentrations of FSH and
LH used were based on previous experiments [10 (link), 11 (link)]. The effect of P4 on follicle growth was evaluated after adding P4 (10 ng/ml,
100 ng/ml, and 1 μg/ml) (Sigma-Aldrich) to the culture medium, followed by culture for 18 days. In this experiment, one ovary was cut into four slices; two of these slices were treated with dimethyl sulfoxide (DMSO) alone and the
other two, with P4 dissolved in DMSO. Four or five ovaries were cultured with each concentration of P4.
placed in a 30-mm cell culture insert (Merck Millipore, Darmstadt, Germany), which was subsequently placed in a 3.5-cm culture dish (AGC Techno Glass, Shizuoka, Japan). Ovarian slices were cultured in the minimum essential medium
alpha (MEM-alpha) GlutaMax (Gibco, Carlsbad, CA, USA) supplemented with 5% (v/v) fetal bovine serum (FBS, Gibco), 100 mIU/ml FSH from human pituitary gland (Sigma-Aldrich, St Louis, MO, USA), and 10 mIU/ml LH from equine pituitary
gland (Sigma-Aldrich) under conditions of 5% CO2 and 37°C. The cultured ovarian tissue slices were treated with 100 mIU/ml of LH for 12 h every 4 days to reproduce the physiological LH surge. Concentrations of FSH and
LH used were based on previous experiments [10 (link), 11 (link)]. The effect of P4 on follicle growth was evaluated after adding P4 (10 ng/ml,
100 ng/ml, and 1 μg/ml) (Sigma-Aldrich) to the culture medium, followed by culture for 18 days. In this experiment, one ovary was cut into four slices; two of these slices were treated with dimethyl sulfoxide (DMSO) alone and the
other two, with P4 dissolved in DMSO. Four or five ovaries were cultured with each concentration of P4.
7
Cell Line Cultivation and Epigenetic Inhibition
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hTERT‐HME1 cells were grown in Cascade Biologics Medium 171 (Gibco). MDA‐MB‐231 cells were grown in Leibovitz's (L‐15) + GlutaMAX Medium (Gibco) supplemented with 10% foetal bovine serum (FBS). MCF‐7 cells were grown in Minimum Essential Medium (MEM) Alpha + GlutaMAX (Gibco), supplemented with Mammary Epithelial Growth Supplement (MEGS) (Gibco, S0155), 10% FBS and 0.01mg/ml human recombinant insulin. All media were passed through vacuum filtration and were protected from contamination by adding 1X Anti‐Anti (antibiotic‐antimycotic) (Gibco). Cells were grown at 37°C at 5% CO2 in T‐75 and T‐150 vent flasks. Media was changed routinely, and cells were passaged using 0.25% Trypsin‐EDTA (1X) (Gibco). To respectively inhibit EZH2/1 or EZH2 methyltransferase function, 5μM UNC1999 (Cell Signaling Technology) or 5 μmol/L tazemetostat (Selleck Chemicals) were prepared in cell line‐specific media and administered to 80%‐90% confluent flasks for 48 hours. BAY11‐7082 inhibitor compound prepared in respective cell media (5 μmol/L) was administered for 24 hours to diminish NF‐κB activity. NKILA was inhibited by transfection of microRNA 103 using Lipofectamine RNAiMAX Reagent, per manufacturer protocol.
8
Isolation and Culture of Amniotic Fluid Stem Cells
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Collection of amniotic fluid and derived products was approved by the Institutional Review Board at New York University School of Medicine. Amniotic fluid was collected at the time of scheduled, term cesarean delivery with the patient’s informed consent (study# i15-01269, New York, NY, USA) and cultured per published protocols to enrich for AFMSCs [13 (link)]. Briefly, amniotic fluid stem cells were isolated from the fluid and cultured in monolayer in medium consisting of 20% Chang medium D (Irvine Scientific, Irvine, CA, USA), MEM-alpha GlutaMAX (Life Technologies, Carlsbad, CA, USA), 15% embryonic stem cell-qualified fetal bovine serum (Life Technologies, Carlsbad, CA, USA) and 100 µg/mL Normocin (InvivoGen, San Diego, CA, USA), then cryopreserved at passage 2. Following thawing, cells were incubated at 37 ℃, 5% CO2, 95% humidity until 80% confluent. We detached cells from the plate using Accutase (Thermo Scientific, Waltham, MA, USA) for further passages or experiments. For the design of this study, AFMSCs from 3 separate batches of amniotic fluid were combined in cell culture.
9
Characterization of Glioblastoma and Colorectal Cell Lines
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The diploid U87-MG cell line contains p53 wild type alleles and the near triploid U251-MG glioblastoma cell line contains mutant p53R273H [49 (link), 50 (link)] and were cultivated as described previously [40 (link)]. The human fetal astroglial SVGp12 cell line (ATCC® CRL8621) was generated by immortalization using of SV40 large T antigen (SV40 TAg) as described earlier [51 (link)] and cultivated using Minimal Eagle Medium (MEM) Alpha Glutamax (Life Technologies, Darmstadt, Germany) supplemented with 10% v/v heat-inactivated FCS (Gibco), 2 mM L-glutamine and 1% non-essential amino acids (Biochrom, Berlin, Germany). The hypodiploid colorectal carcinoma-derived HCT116 cells and HCT116 cells with knockout of p53, designated HCT116p53−/− (kindly provided by B. Vogelstein, Johns Hopkins University, Baltimore) were cultured in RPMI medium (Life Technologies) supplemented with 10% FCS, 2 mM L-glutamine, 10 mM HEPES, 100 U/ml penicillin and 100 μg/ml streptomycin. The human embryonic kidney cells 293 T were maintained in Dulbecco’s modified Eagle medium (DMEM) containing 4.5 g/l glucose (Life Technologies) supplemented with 10% FCS, 10 mM HEPES, 100 U/ml penicillin and 100 μg/ml streptomycin. HCT116, U87-MG, and U251-MG cell lines were authenticated (Multiplexion GmbH, Heidelberg, Germany) and cultured at 37 °C with 5% CO2.
10
Characterization of Human Cell Lines
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The human SS cell lines used in this study have been described previously [26 (link)]. U2OS and 293T cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Human dermal fibroblasts (hDFs) and bone marrow stromal cells (BMSCs) were isolated from donors and maintained in DMEM (4.5 g/l glucose) (Nacalai Tesque, Kyoto, Japan) and MEM Alpha+GlutaMAX (Life Technologies, Carlsbad, CA) supplemented with 10% FBS, respectively. Human embryonic stem cell (hESC) (KhES1) and human induced pluripotent stem cell (hiPSC) (414C2) lines were maintained on SNL feeder cells under previously described culture conditions [25 (link)]. mTeSR1 medium (STEMCELL Technology, Vancouver, Canada) was used for the feeder-free culture of hPSCs.
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