Antibodies against the following proteins were used in this study: Akt, phospho-Akt (Ser473), mTOR, phosphor mTOR, p70 S6 kinase, phospho p70 S6 kinase, STAT3, phospho- STAT3 (Tyr705), and LC3II from Cell Signaling Technology, c-fos, ERK, phospho ERK and β-amyloid (immunohistochemistry) from Santa Cruz Biotechnology, Insulin receptor (Y1158) from Abcam Cambridge, β-amyloid (Immunoblot analysis) from BioLegend, Donkey anti-Mouse IgG from Thermo Fisher Scientific, β-actin from EnoGene, and horseradish peroxidase-conjugated secondary antibodies from Enzo Life Sciences. β-amyloid (human, 1–42) was purchased from Invitrogen. LY294002 and thiazolyl blue tetrazolium bromide (MTT) were purchased from Sigma. PD98059 was purchased from Calbiochem. Recombinant human LIF was purchased from Peprotech. c-fos siRNA was purchased from Santa Cruz Biotechnology.
Recombinant human lif
Manufactured by Thermo Fisher Scientific
Sourced in United States
Recombinant human LIF is a laboratory reagent used in cell culture applications. It is a cytokine that plays a role in the maintenance of embryonic stem cells and the induction of cell differentiation. The core function of this product is to support cell culture research, without further interpretation or extrapolation on its intended use.
Automatically generated - may contain errors
Lab products found in correlation
B27 supplement, by Thermo Fisher Scientific (8 mentions)
Dmem f12, by Thermo Fisher Scientific (6 mentions)
Glutamax, by Thermo Fisher Scientific (6 mentions)
N2 supplement, by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (3 mentions)
Minocycline hydrochloride, by Santa Cruz Biotechnology (2 mentions)
S dimethindene maleate, by Bio-Techne (2 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
Chir99021, by Bio-Techne (2 mentions)
Neurobasal, by Thermo Fisher Scientific (2 mentions)
Neurobasal medium, by Thermo Fisher Scientific (2 mentions)
Lc3 2, by Cell Signaling Technology (1 mentions)
Phosphor mtor, by Cell Signaling Technology (1 mentions)
C fos, by Santa Cruz Biotechnology (1 mentions)
Phospho stat3 tyr705, by Cell Signaling Technology (1 mentions)
Amyloid β, by Santa Cruz Biotechnology (1 mentions)
P70 s6 kinase, by Cell Signaling Technology (1 mentions)
Phospho p70 s6 kinase, by Cell Signaling Technology (1 mentions)
Phospho erk, by Santa Cruz Biotechnology (1 mentions)
7 protocols using recombinant human lif
1
Molecular Signaling Pathways in Neurodegeneration
2
Maintenance of hEPSCs in N2B27-LCDM Medium
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hEPSCs were maintained in an N2B27‐LCDM medium under 20% O2 and 5% CO2 at 37 °C. A total of 500 mL of N2B27‐LCDM medium was prepared, containing 241 mL of DMEM/F12 (Thermo Fisher Scientific, 11330‐032), 241 mL of Neurobasal medium (Thermo Fisher Scientific, 21103‐049), 2.5 mL of N2 supplement (Thermo Fisher Scientific, 17502‐048), 5 mL of B27 supplement (Thermo Fisher Scientific, 12587‐010), 1% nonessential amino acids (Thermo Fisher Scientific, 11140‐050), 0.1 × 10−3m β‐mercaptoethanol (Sigma, M3148), 1% GlutaMAX (Thermo Fisher Scientific, 35050–061), and 5% KnockOut serum replacement (optional) (Thermo Fisher Scientific, A3181502). We added recombinant human LIF (10 ng mL−1; Peprotech, 300–05), CHIR 99021 (1 × 10−6m ; LC Laboratories, C‐6556), IWR‐1‐endo (1 × 10−6m ; Abmole, M2782), DiM (2 × 10−6m ; Tocris, 1425) and MiH (2 × 10−6m ; Tocris, 3268), Y‐27632 (2 × 10−6m ; LC Laboratories, Y‐5301) to the N2B27 medium. hEPSCs were cultured on MEF feeder cells and passaged with 0.05% trypsin for 3 min at 37 °C.
3
Differentiation of human embryonic stem cells into excitatory postsynaptic cells
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hEPSC were cultured and maintained as described[5 ] with minor modifications. Briefly, the cells were cultured on mitomycin‐C (Thermo Fisher Scientific) inactivated STO feeder cells, which were seeded on 0.1% gelatin (Sigma Aldrich)‐coated wells at a density of 0.075 × 106 cells/cm2 at least 2 days prior to hEPSC seeding. The hEPSC medium: DMEM/F12, 1× L‐glutamine, 1× penicillin–streptomycin, 1× NEAA, 0.1 µm 2‐mercaptoethanol, 1× N2 supplement, 1× B27 supplement (Thermo Fisher Scientific), and 65 µg mL−1 L‐ascorbic acid (Sigma Aldrich) supplemented with 2.5 µm XAV939 (Sigma Aldrich), 0.15 µm A419259 (Tocris); 1.0 µm CHIR99021 (Stemgent), 0.25 µm SB590885 (R&D), and 10 ng mL−1 recombinant human LIF (PeproTech). 20% Knock‐out serum replacement (KOSR; Thermo Fisher Scientific) and 10 µm Y‐27632 (Stemcell Technologies) were supplemented to the medium on the day of hEPSC seeding and hEPSC were passaged every 3–4 days.
4
Modulation of Cell Signaling Pathways
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Recombinant human LIF (#300-05, 20 ng/mL), CXCL3 (#300-40, 20 ng/mL) and CXCL8 (#200-08M, 20 ng/mL) were all purchased from Peprotech. LIF neutralizing antibody (AB-250-NA, 2 μg/mL) was purchased from R&D, and CXCL3 neutralizing antibody (PP1014P2, 5 μg/mL) was purchased from Origene. Stattic (#HY-13818), LY3214996 (#HY-101494), PD98059 (#HY-12028), SB 203580 (#HY-10256), LY294002 (#HY-10108), JSH-23 (#HY-13982), BAY 11-7082 (#HY-13453), T-5224 (#HY-12270), TK216 (#HY-122903), TAT-DEF-Elk-1 (#HY-P2262A), SB225002 (#HY-16711), Reparixin (#HY-15251), and EC330 (#HY-100949) were all purchased from Med Chem Express. SB-505124 (#M2250) was purchased from Abmole. A list of the concentrations and targets used for the inhibitors is provided in Supplementary Table 1 .
5
Derivation and Maintenance of EPS Cells
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E3.5 blastocysts were collected from uterus of pregnant NOD-scid Il2rg−/− mice and seeded on mitomycin C (Sigma-Aldrich,M4287)-treated mouse embryonic fibroblast (MEF) feeder cells with LCDM medium containing DMEM/F12 (Thermo Fisher Scientific, 11330-032), Neurobasal (ThermoFisher Scientific, 21103-049), 0.5% N2 supplement (Thermo Fisher Scientific, 17502-048), 1% B27 supplement (Thermo Fisher Scientific, 12587-010), 1% GlutaMAX (Thermo Fisher Scientific, 35050-061), 1% nonessential amino acids (Thermo Fisher Scientific, 11140-050), 1% penicillin-streptomycin (Thermo Fisher Scientific, 15140-122), 0.1 mmol/L β-mercaptoethanol (Thermo FisherScientific, 21985-023), 10 ng/mL recombinant human LIF (Peprotech, 300-05), 3 μmol/L CHIR99021 (Tocris, 4423), 2 μmol/L (S)-(+)-dimethindenemaleate (Tocris, 1425), 2 μmol/L minocycline hydrochloride (Santa Cruz Biotechnology, sc-203339). After 5–8 days, outgrowths were picked up and digested by 0.05% trypsin-EDTA (Thermo Fisher Scientific, 25300-062). The digested single cells were plated on new MEF feeders with LCDM medium. EPS colonies appeared about 4 days later. EPS cells were single-cell passaged every 3 days using 0.05% trypsin-EDTA and used for further characterization.
6
Derivation and Culture of mEPS Cells from Blastocysts
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mEPS cells were derived directly from blastocysts of F1 hybrids between C57 and 129 mice. Blastocysts were cultured on feeder cells for 4–5 days in EPS medium, which contained 120 mL DMEM/F12 (Thermo Fisher Scientific, 11330-032), 120 mL neurobasal (Thermo Fisher Scientific, 21103-049), 1.25 mL N2 supplement (Thermo Fisher Scientific, 17502-048), 2.5 mL B27 supplement (Thermo Fisher Scientific, 12587-010), 1% GlutaMAX (Thermo Fisher Scientific, 35050-061), 1% nonessential amino acids (Thermo Fisher Scientific, 11140-050), 0.1 mmol/L β-mercaptoethanol (Thermo Fisher Scientific, 21985-023), penicillin-streptomycin (Thermo Fisher Scientific, 15140-122), and small molecules and cytokines added to the N2B27 medium at the following final concentrations: 10 ng/mL recombinant human LIF (10 ng/mL; Peprotech, 300-05), CHIR 99021 (3 µmol/L; Tocris, 4423), (S)-(+)-dimethindene maleate (2 µmol/L; Tocris, 1425) and minocycline hydrochloride (2 µmol/L; Santa Cruz Biotechnology, sc-203339). Outgrowths were trypsinized and passaged every 2–3 days for further analysis. EPS-td cells were obtained by EF1α-Tdtomato lentivirus infection.
7
Cell Culture Medium for Stem Cells
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Culture medium was modified Eagle's medium (MEM) supplemented with non-essential amino acids (1×), HEPES (15 mM), gentamicin (40 µg/mL), penicillin/streptomycin (100 U/mL), l-glutamine (4 mM), FBS Australian origin (2.5%), G418 (200 μg/mL), recombinant human basic fibroblast growth factor (bFGF; 2 ng/mL) (Thermo Fisher Scientific), sodium bicarbonate (0.12%), platelet-derived growth factor-BB (10 ng/ mL), forskolin (20 μM), 17β-estradiol (1 nM) (Sigma) and recombinant human LIF (10 ng/mL) (PeproTech, Rocky Hill, USA). VCR sulfate (CAS #2068-78-2) and DXO hydrochloride (CAS #25316-40-9) were obtained from LKT Laboratories Inc (St Paul, MN, USA). Stock solutions of VCR, DXO and the MIX 1:1 were made up at 1000× in sterile water and stored at -80°C. Trypsin/EDTA 0.25% was from Thermo Fisher Scientific (#25200-056).
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