pBOB-NepX or pLKO.1 expression plasmids, and the packaging vectors, pSPAX2 and pMD2.G (Addgene plasmids 12260 and 12259, respectively; both deposited by Dr. Didier Trono, École Polytechnique Fédérale de Lausanne) were transfected using Lipofectamine 2000 (Life Technologies) into HEK293T cells grown in 15 cm Petri dishes to ∼80% confluence in Dulbecco's MEM (Invitrogen) supplemented with 10% HyClone cosmic calf serum (GE Healthcare). Each transfection was with 30 μg total DNA at a 50%/37.5%/12.5% ratio of expression vector/pSPAX2/pMD2.G. Transfection medium was replaced with fresh medium after 24 hours, and lentivirus-conditioned medium was collected 48 and 72 hours after the start of transfection. Lentiviral particles were concentrated in a Beckman Coulter Optima LE-80K ultracentrifuge for 2 hrs at 23,000 rpm (95,000 gav) at 4° C in an SW28 rotor, resuspended in 400 μl Neurobasal medium and stored at −80° C in 20 μl aliquots. Cultured neurons were infected with a viral MOI of 5 and incubated for 24 hours at 37° C, after which lentiviral medium was replaced with fresh Neurobasal plus B27.
Hyclone cosmic calf serum
Manufactured by GE Healthcare
HyClone Cosmic Calf Serum is a laboratory reagent used as a growth supplement in cell culture media. It is derived from the blood of newborn calves. The serum provides a complex mix of nutrients, growth factors, and other essential components to support the growth and proliferation of a variety of cell lines in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Optima le 80k, by Beckman Coulter (3 mentions)
Pspax2, by Addgene (3 mentions)
Pmd2 g, by Addgene (3 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Bob nepx, by Addgene (2 mentions)
Plko 1, by Addgene (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Dulbecco s mem, by Thermo Fisher Scientific (1 mentions)
Matrigel, by Corning (1 mentions)
Gentamycin, by Thermo Fisher Scientific (1 mentions)
Hydrocortisone, by Merck Group (1 mentions)
Sodium selenite, by Merck Group (1 mentions)
Transferrin, by Merck Group (1 mentions)
Sodium hydroxide (naoh), by Fujifilm (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
Hank s balanced salt solution, by Merck Group (1 mentions)
4 protocols using hyclone cosmic calf serum
1
Lentiviral Transduction of Cultured Neurons
2
Isolation and Culture of Colonic Epithelial Cells
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Colons were obtained from mice fed ad libitum and from mice after 36 h fasting. ECs were isolated from the colon and treated with 10 mM EDTA in Hank’s Balanced Salt Solution (Sigma Aldrich). Forty thousand cells/well in a 96-well half plate were cultured in 20% Matrigel (Corning, Tokyo, Japan) in a culture medium containing D-MEM/Media 199 (mixture of 4:1, Sigma and Lonza, Tokyo, Japan), 0.01 M HEPES, 1 × antibiotic-antimycotic (Gibco), 50 µg/ml gentamycin (Gibco) supplemented with 10% Hyclone Cosmic Calf serum (GE Healthcare), 50 ng/ml mEGF (Peprotech, Rocky Hill, NJ), 1 µg/ml hydrocortisone (Sigma-Aldrich), 2 µg/ml Transferrin (Sigma-Aldrich), and 5 nM sodium selenite (Sigma-Aldrich) in 5% CO2 and 5% O2 atmosphere. To study the effects of organic acids, lactate, butyrate, or acetate (10 mM at final concentration, pH was adjusted to 7 with NaOH, Wako, Japan) was added to the culture medium. Fresh medium (0.05 ml) with/without organic acids was added to the culture every 4 days (day 4, 8 and 12). After day 4 of culture, the number of colonies containing more than 10 living cells/well was counted everyday with a inverted microscope (IX71, Olympus, Tokyo, Japan). Cultures were performed in triplicate and data were presented as the mean ± SD, unless otherwise indicated in the legend.
3
Lentiviral Transduction of Mouse Cortical Neurons
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Lentiviral particles for shRNA knockdowns and hTau 2N4R and 0N4R protein expression in mouse cortical neurons were prepared as follows. The expression plasmids, pCSC-SP-PW-NepX (also known as BOB-NepX) or pLKO.1 (Addgene plasmids 12,340 and 10,878, respectively), and the packaging vectors, pSPAX2 and pMD2.G (Addgene plasmids 12,260 and 12,259, respectively) were transfected using Lipofectamine 3000 (ThermoFisher) into HEK293T cells grown in 15 cm Petri dishes to ~80% confluence in DMEM (GIBCO) supplemented with 10% HyClone cosmic calf serum (GE Healthcare). Each transfection was with 15 μg total DNA at a 50%/37.5%/12.5% ratio of expression vector/pSPAX2/pMD2.G. Lentivirus-conditioned medium was collected 24 and 48 h after the start of transfection. Lentiviral particles were concentrated in a Beckman Coulter Optima LE-80 K ultracentrifuge for 2 h at 23,000 rpm (95,000 gav) at 4 °C in an SW28 rotor, resuspended in 400 μl Neurobasal medium and stored at −80 °C in 20 μl aliquots. Cultured neurons were transduced in Neurobasal/B27 medium and incubated for 72 h before assays were performed.
4
Lentiviral Transduction of Mouse Cortical Neurons
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