Hippocampal neurons were plated on poly-ornithine-coated coverslips, transfected 6-8 days after plating, and imaged 14-21 days after plating as previously described (Ryan, 1999 (link)). Neurons were maintained in culture media composed of MEM (Thermo Fisher Scientific 51200038), 0.6% glucose, 0.1 g/L bovine transferrin (Millipore 616420), 0.25 g/L insulin, 0.3 g/L GlutaMAX, 5% fetal bovine serum (Atlanta Biologicals S11510), 2% B-27 (Thermo Fisher Scientific 17504-044), and 4 μm cytosine β-d-arabinofuranoside. Cultures were incubated at 37C in a 95% air/5% CO2 humidified incubator for 14–21 days prior to use.
Bovine transferrin
Manufactured by Merck Group
Sourced in United States
Bovine transferrin is a protein found in the blood serum of cattle. It is responsible for the transport of iron within the body. Bovine transferrin is commonly used in cell culture and other laboratory applications that require a source of iron.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Atlanta Biologicals (3 mentions)
S1200038, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Glutamax supplement, by Thermo Fisher Scientific (1 mentions)
Ar008, by R&D Systems (1 mentions)
Minimum essential medium (mem), by Thermo Fisher Scientific (1 mentions)
Porcine insulin, by Merck Group (1 mentions)
Bovine serum albumin fraction 5, by Merck Group (1 mentions)
Pgf2α, by Merck Group (1 mentions)
Bovine hemoglobin, by Merck Group (1 mentions)
Glass microscope slide, by Avantor (1 mentions)
Conductive indium tin oxide coated glass slides, by Bruker (1 mentions)
Sinapic acid, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Formic acid, by Thermo Fisher Scientific (1 mentions)
Acetic acid, by Thermo Fisher Scientific (1 mentions)
Human serum albumin (hsa), by Merck Group (1 mentions)
α cyano 4 hydroxycinnamic acid, by Merck Group (1 mentions)
Milli q reference system, by Merck Group (1 mentions)
Bradykinin, by Bachem (1 mentions)
12 protocols using bovine transferrin
1
Culturing Hippocampal Neurons for Imaging
2
Isolation and Culture of Hippocampal Neurons
3
Culturing Hippocampal Neurons from Rat Pups
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Hippocampal CA1 to CA3 neurons were isolated from 1- to 3-day-old rats of mixed gender and plated on poly-l -ornithine–coated coverslips as previously described (32 (link)). Calcium phosphate–mediated gene transfer was used to transfect 6- to 8-day-old cultures as described previously (33 (link)). Neurons were maintained in culture media composed of minimum essential medium (Thermo Fisher Scientific, S1200038), 0.6% glucose, bovine transferrin (0.1 g/liter; Millipore, 616420), insulin (0.25 g/liter), glutamine (0.3 g/liter), 5 to 10% fetal bovine serum (BSA) (Atlanta Biologicals, S11510), 2% B-27 (Thermo Fisher Scientific, 17504-044), and 4 mM cytosine b-d -arabinofuranoside. Cultures were incubated at 37°C in a 95% air/5% CO2 in a humidified incubator for 14 to 21 days before use.
4
Culturing Rat Hippocampal Neurons
5
Culturing Hippocampal and Cortical Neurons
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Hippocampi were dissected from 1- to 3-d-old rat pups of mixed sex from the Sprague–Dawley strain. Tissues were dissociated and mixed cultures of neurons and glia were plated on coverslips coated with polyornithine and transfected 6–8 d after plating with calcium phosphate as previously described (Ryan, 1999 (link)). Hippocampal neurons were maintained in culture media composed of MEM (51200038; Thermo Fisher Scientific), 0.6% glucose, 0.1 g/l bovine transferrin (616420; Millipore), 0.25 g/l insulin, 0.3 g/l GlutaMAX supplement (35050-061; Thermo Fisher Scientific), 5% fetal bovine serum (S11510; R&D Systems), 2% N-21 (AR008; R&D Systems), and 4 μm cytosine β-d-arabinofuranoside (Ara-C), added after 2–3 d in vitro (DIV) to limit glial proliferation. Cortical neurons were maintained in Neurobasal-A media (NB-A; # A24775-01; Gibco) supplemented with 2% N-21, 5% fetal bovine serum, and 4 μm Ara-C (added after 1 DIV). Cultures were incubated at 37°C in a 95% air and 5% CO2 humidified incubator for 10–14 DIV (cortical neurons) or 14–21 DIV (hippocampal neurons) prior to use.
6
Hippocampal Neuron Culture Protocol
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Bilateral hippocampi were dissected from postnatal rats or mice (P0–P1; 0–1 d old, both sexes) and plated on poly-ornithine-coated coverslips. Neurons were maintained in culture medium containing of MEM (Thermofisher Scientific, S1200038), 30 mm glucose, 0.1 g/l bovine transferrin (Millipore, 616420), 0.25 g/l insulin, 0.3 g/l glutamine, 5–10% fetal bovine serum (Atlanta Biologicals, S11510), 2% B-27 (Thermofisher Scientific, 17504-044). Cultures were incubated at 37°C with 95% air/5% CO2 in a humidified incubator before imaging. Transfection was performed on day 6 or 7 in vitro (DIV6, DIV7) using Ca2+ phosphate-mediated gene transfer to transfect a low percentage of cells. Live-cell imaging was performed on DIV14–DIV19. For each experiment, neurons were derived from at least three separate culture preparations to minimize artifacts from small variations in culture conditions. The N in each figure corresponds to the number of cells recorded per treatment group.
7
Hormones and Pituitary Compounds Protocol
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The National Hormones and Pituitary Program kindly provided the oLH (NIH-oLH-S26, NIDDK) and (ovine follicle stimulating hormone (oFSH) (NIH-oFSH-S20, NIDDK). P4, T, E2, E, NE, PGF2α, PGE1, corticosterone (B), bovine transferrin, porcine insulin and bovine serum albumin (BSA, fraction V) were purchased from sigma Chemical Co. (St. Louis, MO, USA). cVIP was obtained from Peninsula Laboratories, Inc. (Belmont, CA, USA). The AVT and MT were purchased from Biochemist Inc. (Bubendorf, Switzerland). Antisera to P4 and T were the generous gift of RIA Center of Gunma University. (1, 2, 6, 7-3H) P4, (1, 2, 6, 7-3H) T and ASC-II scintillators were obtained from Amersham International plc. (Buckinghamshire, UK). Mc Coy’s 5a medium without serum and Ham’s F12 medium were obtained from Gibco Life Technologies Inc. (Grand Island, NY, USA). Fetal calf serum was purchased from Boëhringer Mannheim (Mannheim, Germany).
8
Iron Quantification in Metalloproteins
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ACCM-2 containing 5, 25, or 50 μM FeSO4 was prepared in a total volume of 1 ml. Lyophilized powders were used to generate 1-ml aliquots of 50 and 1,250 μg ml−1 bovine hemoglobin (Sigma-Aldrich, St. Louis, MO), bovine transferrin (Sigma-Aldrich, St. Louis, MO), and equine spleen ferritin (Sigma-Aldrich, St. Louis, MO) in MΩH2O. To extract iron, 0.1 ml concentrated HNO3 was added to each sample and incubated at room temperature overnight. Samples were then heated to 80°C for 1 h before being diluted to 5 ml in ddH2O, generating final concentrations of 1, 5, and 10 μM FeSO4 for ACCM-2 and APCM, and 10 and 250 μg ml−1 for each iron-binding protein. Iron content was measured as described above by ICP-MS.
9
MALDI-TOF MS Protein and Peptide Analysis
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Methanol (MeOH), acetonitrile (ACN), acetic acid (AA), 2-nitrobenzaldehyde (NBA), and formic acid (FA) were purchased from Fisher Scientific (Pittsburgh, PA). The matrices of sinapic acid (SA) and α-cyano-4-hydroxycinnamic acid (CHCA) were purchased from Sigma-Aldrich (St. Louis, MO). Microscope glass slides were purchased from VWR international, LLC (Radnor, PA). Conductive indium tin oxide (ITO) coated glass slides were purchased from Bruker (Billerica, MA). Human insulin was purchased from Promega (Madison, WI, USA). Other protein samples (including cytochrome c from bovine heart, bovine serum albumin, human serum albumin and bovine transferrin) were purchased from Sigma-Aldrich (St. Louis, MO). Peptide standards (bradykinin, somatostatin II, angiotensin II, FMRF, FMRFamide-like peptide II (lobster)) were purchased from American Peptide Company (Sunnyvale, CA, USA). Amyloid beta peptides were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). No further purifications were performed for all reagents. All solvents used in this study were of HPLC grade. Purified water (conductivity of 18.2 MΩ.cm) was obtained from Milli-Q Reference System (Millipore Corp., Bedford, MA, USA).
10
Hippocampal Neuron Culture from Neonatal Mice
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Hippocampus of postnatal day 0 C57BL/6J pups were dissected under the microscope and cut into pieces. Then, the tissue was digested by trypsin bathed with 37°C water for 5 min and suspended. After 5 min centrifugation, 100 µL plating medium containing cells was plated on 12 mm coverslips coated with matrix gel (Corning) in 35 mm dishes. Two microliters of plating medium was added to the dishes 2 h later. A hundred milliliters of plating medium consist of 89 mL Minimal Essential Medium (Invitrogen), 10 mL fetal calf serum, 0.5 mM glutamine, 2 g NaHCO3, 0.5 g glucose, 10 mg bovine transferrin (Calbiochem), and 2.5 mg insulin. Forty‐eight hours after the plating, half of the medium was replaced by feeding medium for twice a week. A hundred microliters of feeding medium consist of 97 mL Minimal Essential Medium (Invitrogen), 2 mL B27 medium supplement (Invitrogen), 10 mg bovine transferrin, 0.5 mM glutamine, 0.5 g glucose, 3 mM cytosine‐p‐arabinofuranoside (Sigma–Aldrich), and 2 g NaHCO3.
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