Cell migration assays were performed and analyzed with an IncuCyte S3 Life Cell Imaging System (EssenBio Science). Briefly, U87 cells were harvested and resuspended in Minimum Essential medium (Biowest) supplemented with 0.5% (v/v) heat-inactivated FCS (Pan Biotech), 1 unit/ml penicillin-streptomycin (Biowest), 2 mM L-glutamine (Biowest), and 1 unit/ml non-essential amino acids (Biowest). Approximately, 1000 cells were placed into the top chamber of each well of a 96-well ClearView Plate (EssenBioscience). Chemoattractants were mixed in 200 μl medium and then placed in the corresponding bottom chambers. Image acquisition was performed hourly for 24 h on both sides of a membrane separating the top and bottom chambers. Subsequent analysis was performed using the IncuCyte S3 software.
Minimum essential medium (mem)
Manufactured by Biowest
Sourced in France, United States, Germany
MEM is a cell culture medium that provides essential nutrients and growth factors for the maintenance and proliferation of various cell lines in vitro. It is a widely used and versatile media formulation suitable for a range of cell types.
Automatically generated - may contain errors
Lab products found in correlation
L glutamine, by Biowest (7 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Mcf 7, by American Type Culture Collection (4 mentions)
Fetal bovine serum (fbs), by Biowest (4 mentions)
Antibiotic antimycotic, by Biowest (3 mentions)
Fetal bovine serum (fbs), by Harvard Bioscience (3 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin p s, by Thermo Fisher Scientific (3 mentions)
Mg 63, by American Type Culture Collection (3 mentions)
Transferrin, by Merck Group (3 mentions)
Insulin, by Sanofi (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Merck Group (3 mentions)
Penicillin streptomycin, by Biowest (2 mentions)
Hcc1806, by American Type Culture Collection (2 mentions)
Pcr mycoplasma test kit 1 c, by PromoCell (2 mentions)
Mda mb 231, by American Type Culture Collection (2 mentions)
Fetal calf serum (fcs), by PAN Biotech (1 mentions)
Non essential amino acids (neaa), by Biowest (1 mentions)
Fetal calf serum (fcs), by Biowest (1 mentions)
22 protocols using minimum essential medium (mem)
1
Cell Migration Assay using IncuCyte
2
Treating Fibroblasts with Antisense Oligonucleotides
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Human dermal fibroblasts from patient and controls were cultured in Minimum Essential Medium (Biowest, Renningen, Germany), containing 20% fetal calf serum (Biowest), 1.4% L-glutamine (Biowest), and 1% Antibiotic-Antimycotic (Biowest) at 37°C and 5% CO2. One day before treatment, 1.8–2.5 × 105 cells per well were seeded either in a twelve-well or six-well plate. One of six AONs was added directly to the medium without using any transfection reagent in different concentrations. Concentrations varied between 200 nM, 36 nM, 20 nM, 18 nM, and 9 nM. After 20–96 h of incubation at 37°C, the cells were washed with 1 mL of 1−× phosphate-buffered saline (Biowest), lyzed with 350 μl of lysis buffer RA1 (Macherey and Nagel, Düren, Germany), and supplemented with 3.5 μl of β-Mercaptoethanol (β-ME; Serva, Heidelberg, Germany). Lysates were harvested by pipetting up and down several times, as well as by scraping with a pipette tip.
3
Donor Cornea Preparation for DSAEK and UT-DSAEK
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Donor corneas were precut by a single cornea bank (Euro Tissue Bank, Beverwijk, the Netherlands). Donor tissue was preserved according to conventional eye-bank techniques. The selection criteria of the donor cornea for DSAEK or UT-DSAEK were the same. After a short hypothermic storage between recovery and arrival at the bank, corneoscleral buttons were dissected and stored in organ culture comprising minimum essential medium (Biowest, Nuaillé, France) supplemented with 25 mmol/l 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 26 mmol/l sodium bicarbonate, 5.5 mmol/l glucose, 2 mmol/l L-glutamine, 1 mmol/l pyruvate, 2% (vol/vol) newborn calf serum, 10 IU/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 mg/ml amphotericin at 31 C. To allow deturgescence, corneoscleral buttons were transferred to a transport medium supplemented with 6% dextran (Sigma Aldrich, St. Louis, MO) before and immediately after dissection. Graft dissection was performed with the Gebauer SLc microkeratome system (Gebauer Medizintechniek GmbH, Neuhausen, Germany) using a single-pass technique, 12, 13 aiming at a central residual stromal bed thickness of 200AE20 mm for DSAEK and 100AE20 mm for UT-DSAEK. Donor and lamellar central corneal thickness were measured at the cornea bank using anterior-segment optical coherence tomography (AS-OCT; Cassia SS-1000; Tomey, Nagoya, Japan).
4
Hep-2 Cell Culture Protocol
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Hep-2 cells purchased from the Chinese Type Culture Collection (CTCC; Wuhan,
China) were maintained in the following culture medium: Minimum essential medium
(MEM; BioWest, Loire Valley, France) supplemented with 10% foetal bovine serum
(FBS; Gibco, ThermoFisher Scientific, Waltham, MA, USA), 100 U/ml penicillin G,
and 100 μg/ml streptomycin at 37°C in a humidified atmosphere of 95% air and 5%
CO2. CpG ODN7909 (5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′) was obtained
from Shanghai Sangon Biological Engineering Technology and Services Limited
Company (Sangon, Shanghai, China), dissolved in phosphate buffer saline (PBS;
0.01 M, pH 7.4) and maintained at –20°C until use.
China) were maintained in the following culture medium: Minimum essential medium
(MEM; BioWest, Loire Valley, France) supplemented with 10% foetal bovine serum
(FBS; Gibco, ThermoFisher Scientific, Waltham, MA, USA), 100 U/ml penicillin G,
and 100 μg/ml streptomycin at 37°C in a humidified atmosphere of 95% air and 5%
CO2. CpG ODN7909 (5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′) was obtained
from Shanghai Sangon Biological Engineering Technology and Services Limited
Company (Sangon, Shanghai, China), dissolved in phosphate buffer saline (PBS;
0.01 M, pH 7.4) and maintained at –20°C until use.
5
Fibroblast Isolation and Cilia Analysis
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Skin biopsies were obtained from the index patient and two unaffected controls. The skin biopsies were cut into smaller pieces and transferred to a sterile culture flask. After the explants were attached to the bottom of the flask by air drying, minimal essential medium (MEM, Biowest, Nuaille, France) supplemented with 1.3% L-glutamine, 0.8% antibiotic and 20% fetal bovine serum (FBS) was added and incubated at 37 °C and 5% CO2. Fibroblasts growing out from the explants were harvested and transferred to 75 cm2 flaks for propagation and analyses. Cells were passaged when 80–90% confluency was reached. To inhibit NMD, cells were incubated with 100 µg/mL cycloheximide (Sigma-Aldrich, St. Louis, MO, USA) for 4 h at 37 °C and 5% CO2. For immunostaining 0.1 × 106 fibroblast cells were seeded in 12-well plates and cultured for 24 h in normal MEM medium or in MEM containing 30 µM PTC124. Cilia generation was induced by starving the cells for 48h in FBS-free MEM with or without PTC124.
6
Culturing Human Colon Cancer Cell Lines
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The human colon carcinoma CEA-positive, onalespib-sensitive cell line HT55 was obtained from the European Collection of Authenticated Cell Culture (ECACC [43 (link)]), and the colon adenocarcinoma CEA-positive, onalespib-sensitive cell line SNU1544 was obtained from Korean Cell Line Bank (KCLB [44 (link),45 (link)]). CEA expression level and onalespib sensitivity in both cell lines have been previously assessed [27 (link),46 (link)]. The HT55 cells were cultured in Minimum Essential Medium (MEM) (Biowest, Riverside, MO, USA) supplemented with 20% (v/v) fetal bovine serum (FBS; Sigma Aldrich, St. Louis, MO, USA). The SNU1544 cell line was cultured in RPMI [(Biowest, MO, USA, containing 25 nM N-2-Hydroxyethylpiperazine-N’-2-Ethanesulfonic Acid (HEPES)] supplemented with 10% (v/v) heat-inactivated FBS (Sigma Aldrich, MO, USA). Heat inactivation was done at 56 °C for 30 min. All media were supplemented with antibiotics (100 IU penicillin and 100 µg/mL streptomycin, Biochrom GmbH, Berlin, Germany) and L-glutamine (Biochrom GmbH, Berlin, Germany, 2 mM). Monolayer cultures were grown in tissue culture flasks (VWR, Radnor, PA, USA) and incubated in an atmosphere containing 5% CO2 at 37 °C. After reaching 75–85% confluency, cell passaging was performed using Trypsin-EDTA (Biochrom GmbH, Germany). All cell lines were cultured less than 3 months after purchase.
7
Culture and Maintenance of Liver Cell Lines
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All cell lines were obtained from the American Type Culture Collection apart from the immortalized human hepatocytes IHH cell line that was provided by the European Genomic Institute for Diabetes (Lille). The human liver cancer HepG2 cell line was cultured in Dulbecco's Modified Eagle's Medium (DMEM; Lonza Group Ltd.) supplemented with 25 mM glucose. The human hepatocarcinoma Hep3B cell line was cultured in Minimal Essential Medium (MEM; Biowest SAS) supplemented with 5 mM glucose. The immortalized human hepatocyte IHH cell line was cultured in William's E Medium (Lonza Group Ltd.) supplemented with 10 mM glucose. All cells were maintained in medium supplemented with 10% (v/v) fetal calf serum (Dominique Dutscher SAS) and 2 mM L-glutamine and incubated at 37°C in a 5% (v/v)CO2-enriched humidified atmosphere. To maintain optimal growth conditions, cells were divided before confluence was reached and fresh medium was added. The day before cell were used, the cells were divided to retain their ability to proliferate.
8
Cell Culture Protocols for Human Cell Lines
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Experiments were carried out on four human cell lines: keratinocyte—HaCaT (purchased from ThermoFisher (Waltham, MA, USA)), malignant melanoma—A375 (purchased from ATCC (Manassas, VA, USA)), breast adenocarcinoma—MCF-7 (purchased from ATCC (Manassas, VA, USA)), and lung carcinoma—A549 (purchased from ATCC (Manassas, VA, USA)). The culture medium for HaCaT, A375, and MCF-7 cells was based on Dulbecco’s modified Eagle’s medium (DMEM, Biowest (Nuaille, France)), supplemented with 10% of fetal bovine serum (FBS, Biowest (Nuaille, France)), 1% 25 mM l -glutamine (Biowest (Nuaille, France)), 1% 100 mM penicillin, and streptomycin (Biowest (Nuaille, France)). The culture medium for A549 differed only in the base; it consisted of minimum essential medium (MEM, Biowest (Nuaille, France)) with the same additives as before. The cells were sub-cultured every two days using Tryple Express (Gibco (Waltham, MA, USA)) solution to detachment. Cells were kept at 37 °C and 5% CO2 in a humidified atmosphere.
9
Colon and Cervical Cancer Cell Treatment
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The human colon cancer cells (Caco-2) were grown in Minimum Essential Medium (MEM), obtained from BIOWEST (BIOWEST, Riverside, CA, USA), supplemented with 20% fetal bovine serum (FBS), 100 i.u./mL penicillin, 100 µg/mL streptomycin at 37 °C in 5% CO2. Alternatively, human cervical cancer cells (HeLa) were grown in MEM supplemented with 10% fetal bovine serum (FBS), 100 i.u./mL penicillin, 100 µg/mL streptomycin at 37 °C in 5% CO2. Cells were left under basal condition or treated for 2 h with 300 mg/L GAE of non-alcoholic extracts of NA, NT, N, P, and SM cultivars. Three biological replicates were considered in this analysis too.
10
Culturing Vero Cells for PEDV Study
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African green monkey kidney cell lines (Vero) were cultured in Minimum Essential Media (MEM; biowest, Nuaillé, France) supplemented with 10 mM HEPES (Gibco, Carlsbad, CA, USA) and 10% antibiotic-antimycotic (Gibco, Carlsbad, CA, USA) fetal bovine serum (Gibco, Carlsbad, CA, USA) at 37 °C with 5% CO2. In this study, we used the PEDV strain PED-CUP-B2014 [30 (link)]. Virus titer was determined by TCID50 assay [31 (link)].
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