The radiotherapy-resistant oral cavity cancer cell line (UPCI-SCC-131) and fibroblast cell line (NIH-3T3) were purchased from DSMZ (Braunschweig, Germany) and ATCC (American Type Culture Collection CRL-1658, Manassas, VA, USA), respectively. Cells were cultured with minimum essential medium (MEM) (Biochrom, Cambridge, UK) and supplemented with 10% fetal bovine serum (FBS) (Biochrom, Cambridge, UK), 2 mM L-Glutamine (Gibco, Grand Island, NY), and penicillin (100 units/mL)/streptomycin (100μg/mL) (Gibco, Grand Island, NY) and were maintained in a humidified incubator (5% CO2 at 37°C).
Minimum essential medium (mem)
Manufactured by Harvard Bioscience
Sourced in Germany, United Kingdom
MEM is a laboratory equipment designed for cell culture and tissue engineering applications. It is used to maintain and support the growth of various cell types in controlled in vitro environments.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Non essential amino acids (neaa), by Harvard Bioscience (4 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Harvard Bioscience (3 mentions)
Propidium iodide, by Thermo Fisher Scientific (3 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (3 mentions)
Metafectene, by Thermo Fisher Scientific (2 mentions)
Fetal calf serum (fcs), by Harvard Bioscience (2 mentions)
L glutamine, by Harvard Bioscience (2 mentions)
Penicillin streptomycin, by Harvard Bioscience (2 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Carboxymethylcellulose, by Merck Group (2 mentions)
Nih 3t3, by Leibniz Institute DSMZ (1 mentions)
Fuchsin substrate chromogen, by Agilent Technologies (1 mentions)
Hepes buffer, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Amphotericin b, by Thermo Fisher Scientific (1 mentions)
L ascorbic acid, by Merck Group (1 mentions)
22 protocols using minimum essential medium (mem)
1
Culturing Radiotherapy-Resistant Oral Cancer Cells
2
Isolation and Culture of Human Primary Osteoblasts
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The isolation of the human primary osteoblasts (hOB) was performed according to a previously described protocol under sterile conditions [20 (link)]. The human primary osteoblasts were taken from the spongiosa of the femoral heads of patients who underwent total hip replacement. The study was approved by the Local Ethical Committee of Rostock, Germany (registration number: A2010-10), and an informed consent was signed by each patient.
The human osteoblasts were cultivated in modified Eagle’s osteogenic cell culture medium (MEM; Biochrom AG, Berlin, Germany) containing 10% fetal calf serum (FCS), 1% penicillin/streptomycin, 1% amphotericin B, and 1% HEPES buffer (all from Gibco-Invitrogen, Darmstadt, Germany) without calcium, and the osteogenic additives dexamethasone (100 mM), L-ascorbic acid (50 μg/mL), and β-glycerophosphate (10 mM) (all from Sigma-Aldrich, Munich, Germany). The osteogenic differentiation of the human primary osteoblasts was confirmed by immunohistochemical detection of the enzyme alkaline phosphatase using a fuchsin+substrate chromogen (DAKO, Hamburg, Germany). For the further tests, the isolated cells were cultured in 25-cm2 flasks with 8 ml of osteoblasts in the same medium but without the 1% penicillin/streptomycin and under standard cell culture conditions (5% CO2 and 37°C).
The human osteoblasts were cultivated in modified Eagle’s osteogenic cell culture medium (MEM; Biochrom AG, Berlin, Germany) containing 10% fetal calf serum (FCS), 1% penicillin/streptomycin, 1% amphotericin B, and 1% HEPES buffer (all from Gibco-Invitrogen, Darmstadt, Germany) without calcium, and the osteogenic additives dexamethasone (100 mM), L-ascorbic acid (50 μg/mL), and β-glycerophosphate (10 mM) (all from Sigma-Aldrich, Munich, Germany). The osteogenic differentiation of the human primary osteoblasts was confirmed by immunohistochemical detection of the enzyme alkaline phosphatase using a fuchsin+substrate chromogen (DAKO, Hamburg, Germany). For the further tests, the isolated cells were cultured in 25-cm2 flasks with 8 ml of osteoblasts in the same medium but without the 1% penicillin/streptomycin and under standard cell culture conditions (5% CO2 and 37°C).
3
Transient Transfection of TAAR1 Variants
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HEK293 cells were maintained in Minimum Essential Media (MEM, Biochrom GmbH, Berlin, Germany) supplemented with 5% fetal calf serum (FCS) and non-essential amino acids (Biochrom AG, Berlin, Germany), in humidified air at 37°C and 5% CO2. Cells were seeded in poly-L-lysine coated (Biochrom GmbH, Berlin, Germany) 96-well assay plates, at a density of 1.5 × 105 cells/ml. After 24 h, cells were transiently transfected using METAFECTENE (0.45 μl/well), in supplement-free Advanced MEM (Life Technologies, Carlsbad, CA, USA). For the HiBiT assay, cells were transfected with the pcDNA3 empty vector (mock), TAAR1-WT, or co-transfected with TAAR1-WT and variants to resemble the heterozygous state, as well as GLPR as a positive control. For the GloSensor™ cAMP assay, cells were co-transfected with TAAR1-WT and variants and the GloSensor plasmid F22 (Promega, Mannheim, Germany). As a negative control, TAAR1 was exchanged with empty vector (mock). In order to mimic the heterozygous state of the variants, TAAR1-WT and mutants were transfected in equimolar plasmid amounts.
4
Isolation of Hippocampal Neurons from Mice
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Hippocampal neurons from E17-E19 C57BL/6 mice brains were prepared according to standard procedures57 (link). Briefly, hippocampal neurons were incubated for 10 min at 37 °C in trypsin-EDTA solution (0.025%/0.01% w/v; Biochrom AG) in 1 × PBS, and tissues were mechanically dissociated at RT with a fire polished Pasteur pipette. Cell suspension was harvested and centrifuged at 600 × g for 5 min. The pellet was resuspended in MEM (Biochrom AG) containing 5% fetal bovine serum, 5% horse serum, 2 mM glutamine, 1% penicillin-streptomycin antibiotic mixture (Sigma-Aldrich), and 25 mM glucose. The cells were plated on poly-l -lysine (0.1 mg mL−1; Sigma-Aldrich) pre-coated wells. After 72 h, medium was replaced again with glutamine-free medium and experiments were performed at 12–14 days in vitro (DIV).
5
Osteoblast Adhesion to Scaffolds
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The adhesion of osteoblast-like MC3T3-E1 to the scaffolds was visualized
by detecting the filamentous actin of the cytoskeleton of cells on
the scaffolds. Live/dead staining was carried out to assess the cytotoxicity
of the scaffolds. In brief, the scaffolds were immersed into MEM (Biochrom,
Germany) to stabilize the pH value prior to the seeding of cells on
the scaffolds. After the pre-treatment, 0.2 mL of MC3T3-E1 cell suspension
(2 × 105 cells/mL) was added on the scaffolds (in
24-well plates). After 3 h of incubation, an additional 1.8 mL of
MEM was added. The culture medium was changed every 2 days. To minimize
the influence of cells adhering to the bottom surface of the well
during cultivation, the scaffolds were placed into new wells of a
24-well-plate when exchanging the medium. After 21 days of culture,
cell adhesion on the scaffolds was visualized by staining. Cell nuclei
were stained by 4,6-diamidino-2-phenylindole (DAPI, dilactate, Invitrogen),
whereas the live/dead assay was carried out using calcein (Thermo
Fisher, Germany) and propidium iodide (Thermo Fisher, Germany), according
to the manufacturer’s protocol. Images of the fluorescently
stained MC3T3-E1 cells were taken by a fluorescence microscope (Axio
Scope A.1, Carl Zeiss Microimaging GmbH, Germany).
by detecting the filamentous actin of the cytoskeleton of cells on
the scaffolds. Live/dead staining was carried out to assess the cytotoxicity
of the scaffolds. In brief, the scaffolds were immersed into MEM (Biochrom,
Germany) to stabilize the pH value prior to the seeding of cells on
the scaffolds. After the pre-treatment, 0.2 mL of MC3T3-E1 cell suspension
(2 × 105 cells/mL) was added on the scaffolds (in
24-well plates). After 3 h of incubation, an additional 1.8 mL of
MEM was added. The culture medium was changed every 2 days. To minimize
the influence of cells adhering to the bottom surface of the well
during cultivation, the scaffolds were placed into new wells of a
24-well-plate when exchanging the medium. After 21 days of culture,
cell adhesion on the scaffolds was visualized by staining. Cell nuclei
were stained by 4,6-diamidino-2-phenylindole (DAPI, dilactate, Invitrogen),
whereas the live/dead assay was carried out using calcein (Thermo
Fisher, Germany) and propidium iodide (Thermo Fisher, Germany), according
to the manufacturer’s protocol. Images of the fluorescently
stained MC3T3-E1 cells were taken by a fluorescence microscope (Axio
Scope A.1, Carl Zeiss Microimaging GmbH, Germany).
6
Cultivation and Purification of Modified Vaccinia Virus
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Test virus suspensions were prepared by infecting susceptible cells with MVA from the Institute of Animal Hygiene and Veterinary Public Health at the University of Leipzig. BHK-21 cells, a cell line established from fibroblasts of newborn hamster kidneys were used for virus cultivation and the suspension test. After virus inoculation of the cells, the supernatant was replaced by minimum essential medium (MEM, Biochrom AG, Germany) with 2 % foetal calf serum (FCS, Sigma-Aldrich, Germany). The host cells (Collection of Cell Lines in Veterinary Medicine [CCLV], Friedrich Loeffler Institute, Germany) were cultivated at 37 °C in a humid atmosphere under 5.0 % CO2. For the virus cultivation, confluent monolayers with a maximum age of 2 days were used. The cells were incubated at 37 °C until 70–95 % of the cells exhibited a cytopathic effect (approximately 7 days). The cells were frozen and thawed twice, followed by centrifugation at 1,900 g for 10 min. The virus titre was up-scaled by ultra centrifugation at 4 °C at 53,900 g for 2.5 h. The pellet was resuspended in 2 mL medium and aliquots of the virus suspension were stored at -70 °C.
7
Cultivation and Characterization of HEK-293 Cells
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The HEK-293 cell line was purchased from ATCC. Cells were cultivated in minimal essential medium (MEM; Biochrom, Berlin, Germany) supplemented with 5% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) and 1% nonessential amino acids (NEAA; Biochrom, Berlin, Germany) at 37 °C in humidified air containing 5% CO2. The HEK-293 cells were tested for mycoplasma contamination at regular intervals. For measurements of cAMP, cell viability, total, and cell surface expression as well as for reporter gene assays, 1.5 · 104 cells per well were seeded in 96-well plates and incubated for 24 h. For viability and reporter gene assays, translucent 96-well plates (Falcon, Kaiserslautern, Germany) coated with poly-L-lysine (Gibco, Waltham, MA, USA) were used, and for cAMP and total and cell surface expression, white 96-well plates (Corning, Costar, NY, USA). For confocal microscopy, 1.5 · 105 cells were seeded in translucent 6-well plates with one round 24 mm glass coverslip, #1.5, added to each well.
8
Cell Culture Protocols for Hepatoma, Kidney Cells
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Human hepatoma cells (HepG2) were cultured
in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen,
Karlsruhe, Germany) supplemented with 10% fetal calf serum (FCS) (GE
Healthcare), 100 μg/mL of streptomycin, 100 IU/mL of penicillin
(Invitrogen), 2 mMl -glutamine and 1% non-essential amino
acids (Invitrogen) at 37 °C in a 5% (v/v) CO2 incubator.
Cells were grown on sterile collagen-coated (SERVA Electrophoresis
GmbH, Heidelberg, Germany) culture plates. Huh7 cells were maintained
in DMEM supplemented with 10% FCS, 2 mMl -glutamine, 0.1
mM non-essential amino acids and 1% penicillin/streptomycin.
African green monkey (Chlorocebus sp.) kidney cells
(Vero E6, Collection of Cell Lines in Veterinary Medicine CCLV, Friedrich-Loeffler-Institut,
Greifswald-Insel Riems, Germany) were grown and maintained in Eagle’s
minimal essential medium (MEM; Biochrom GmbH, Berlin, Germany) supplemented
with 10% FCS (Biochrom GmbH, Berlin, Germany) and kept under a 5%
CO2 atmosphere at 37 °C.
in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen,
Karlsruhe, Germany) supplemented with 10% fetal calf serum (FCS) (GE
Healthcare), 100 μg/mL of streptomycin, 100 IU/mL of penicillin
(Invitrogen), 2 mM
acids (Invitrogen) at 37 °C in a 5% (v/v) CO2 incubator.
Cells were grown on sterile collagen-coated (SERVA Electrophoresis
GmbH, Heidelberg, Germany) culture plates. Huh7 cells were maintained
in DMEM supplemented with 10% FCS, 2 mM
mM non-essential amino acids and 1% penicillin/streptomycin.
African green monkey (Chlorocebus sp.) kidney cells
(Vero E6, Collection of Cell Lines in Veterinary Medicine CCLV, Friedrich-Loeffler-Institut,
Greifswald-Insel Riems, Germany) were grown and maintained in Eagle’s
minimal essential medium (MEM; Biochrom GmbH, Berlin, Germany) supplemented
with 10% FCS (Biochrom GmbH, Berlin, Germany) and kept under a 5%
CO2 atmosphere at 37 °C.
9
Cell Culture for Infection Studies
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Human lung carcinoma alveolar epithelial (A549) cells were maintained in Dulbecco’s modified Eagle medium (DMEM)-10% [v/v] fetal calf serum (FCS) at 37 °C under 5% CO2 atmosphere. Monkey kidney epithelial (Vero E6) cells were maintained in Biochrom minimum essential media (MEM) with Earle’s salts supplemented with 10% [v/v] FCS, 0.625% l -glutamine, 0.5% penicillin-streptomycin and 0.5% NEAA (Biochrom, Cambridge, United Kingdom) at 37 °C. For infection studies, cells were seeded in 24-, 48- or 96-well plates and cultured 16 h until cell monolayer formation.
10
TNBC Cell Line Expansion and Maintenance
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TNBC cell lines HCC1806, HCC70 and MDA-MB-231 were obtained from American Type Culture Collection (ATCC, Manassas, Virginia, USA). In order to guarantee the identity of the cell lines over the years, cells were expanded after purchase and aliquots were stored in liquid nitrogen. Every half year a new frozen stock was opened and expanded to carry out the experiments. Cells were cultured in MEM (Biochrom, Berlin, Germany) supplemented with 2 mM glutamine, 6 ng/ml insulin, 10 ng/ml transferrin, penicillin (50 U/ml), streptomycin (50 μg/ml) from Gibco (Paisley, UK), and 5% fetal bovine serum (Biochrom, Berlin).
17β-estradiol (E2), estriol, insulin, and transferrin were from Sigma-Aldrich (Deisendorf, Germany). G15 was purchased from R & D systems (Wiesbaden, Germany). Primers for PCR and biotin-labeled oligonucleotide probes for electrophoretic mobility shift assay were produced by MWG-eurofins (Ebersberg, Germany).
17β-estradiol (E2), estriol, insulin, and transferrin were from Sigma-Aldrich (Deisendorf, Germany). G15 was purchased from R & D systems (Wiesbaden, Germany). Primers for PCR and biotin-labeled oligonucleotide probes for electrophoretic mobility shift assay were produced by MWG-eurofins (Ebersberg, Germany).
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