Isoflurane was used at aqueous concentrations equivalent to 0.25–2 times minimum alveolar concentration (MAC) in rat (Taheri et al., 1991 (link)) as a clinically relevant dose range (1 MAC is ED50). Saturated stock solutions were diluted to working concentrations for superfusion through Teflon tubing using gas-tight glass syringes. Coverslips were placed in a heated (37°C) perfusion chamber (260 μl) and Tyrode’s solution [in mM: 119 NaCl, 2.5 KCl, 2 CaCl2, 2 MgCl2, 25 HEPES, and 30 glucose (pH 7.4)] ± isoflurane was delivered using a custom pressurized, inline heated, gas-tight superfusion system (Hemmings et al., 2005 (link)) at 2 ml/min (equilibration time constant of ∼8 s). Cells were equilibrated with control or anesthetic solutions prior to start of experiments and anesthetic concentrations from bath and syringe samples were confirmed by gas chromatography (Herold et al., 2009 (link)). TrkB-Fc (1 μg/ml; ED50 = 0.1–0.4 μg/ml; R and D Systems, Minneapolis, MN) and recombinant BDNF (75–100 ng/ml; PeproTech, Rocky Hill, NJ; Yang et al., 2016 (link)) were added 5 min prior to stimulation. Drug concentrations were based on cell type, stimulation frequency, and incubation times (Rex et al., 2007 (link)).
Recombinant bdnf
Manufactured by Thermo Fisher Scientific
Sourced in United States
Recombinant BDNF is a laboratory-produced protein that replicates the structure and function of naturally occurring brain-derived neurotrophic factor (BDNF). BDNF is a growth factor that supports the survival and differentiation of neurons.
Automatically generated - may contain errors
Lab products found in correlation
Trkb fc, by R&D Systems (1 mentions)
Anti phospho erk, by Cell Signaling Technology (1 mentions)
Rapamycin, by Merck Group (1 mentions)
Anti flag, by Merck Group (1 mentions)
Anti 14 3 3ζ, by Santa Cruz Biotechnology (1 mentions)
Anti map2, by Santa Cruz Biotechnology (1 mentions)
Anti hnrnpa2b1, by Abcam (1 mentions)
Anti fmrp, by Cell Signaling Technology (1 mentions)
Anti glua1, by Abcam (1 mentions)
Anti actin, by MP Biomedicals (1 mentions)
Anti hnrnpd, by Merck Group (1 mentions)
Anti hnrnp q, by Merck Group (1 mentions)
Anti phospho rps6, by Abcam (1 mentions)
Anti hnrnp a1, by Santa Cruz Biotechnology (1 mentions)
Anti psd95, by Abcam (1 mentions)
Ana 12, by Merck Group (1 mentions)
Trkb fc chimera, by R&D Systems (1 mentions)
Dmem high glucose, by Fujifilm (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Collagen coated dishes, by Iwaki (1 mentions)
6 protocols using recombinant bdnf
1
Isoflurane Superfusion of Cultured Cells
2
Investigating Synaptic Protein Regulation
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Antibodies used in this study are as follows: anti-GluA1, anti–hnRNP A2/B1, anti–PSD-95 (Abcam), anti–phospho-RPS6, anti–phospho-ERK (extracellular signal–regulated kinase), anti-FMRP (Cell Signaling), anti–N-term-GluA1, anti–N-term-GluA2, anti–hnRNP D (Millipore), anti-MAP2, anti–hnRNP A1, anti–14-3-3ζ (Santa Cruz), anti–hnRNP Q, anti-Flag (Sigma-Aldrich), anti-NMDAR1 (Synaptic Systems), and anti-actin (MPBIO). To inhibit translation, SHSY5Y cells were treated with 10 nM RAD001 or cycloheximide (100 μg/ml) and harvested at the indicated times. To block cap-dependent translation or induce synaptic stimulation, neurons were treated with 20 nM rapamycin (Sigma-Aldrich) or recombinant BDNF (100 ng/ml) (PeproTech), respectively.
3
Preparation of Protein Reagents for In Vivo Use
4
Hippocampal BDNF Injection in Infant Rats
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PN15 rat pups were anesthetized with isoflurane mixed with oxygen. Stainless steel cannulas (26-gauge) were implanted bilaterally in the dorsal hippocampus (for PN15, 3.0 mm anterior, 2.2 mm lateral and 2.3 mm ventral from bregma) through holes drilled in the overlying skull. The cannulas were fixed to the skull with dental cement. After recovery from the surgery, pups were returned to the dam and littermates. Hippocampal injections used a 33-gauge needle that extended 1 mm beyond the tip of the guide cannula and connected via polyethylene tubing to a Hamilton syringe. Injections were delivered at a rate of 0.1 μL min−1 using an infusion pump on a total volume of 0.3 μL per side over 3 min. The injection needle was left in place for 2 min after the injection to allow complete diffusion of the solution. Recombinant BDNF (PeproTech, cat# 450-02) was dissolved in 1× PBS and injected at 0.25 μg per side. This dose was shown to rescue the infantile amnesia for IA memory in PN17 rats (Travaglia et al. 2016a (link)).
5
Modulation of Myoblast Differentiation by Recombinant Proteins
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Hu5/KD3 cells are a human myoblast cell line [39 (link)]. The cells were cultured on collagen-coated dishes (Iwaki) in high-glucose DMEM (FujiFilm) supplemented with 20% FBS (Gibco) and 2% Ultroser G (Biosepta, Pall) as previously described [39 (link)]. Adult human skeletal muscle myoblasts (hSKMM) were obtained from Lonza (#CC-2580) and cultured on collagen-coated dishes (Iwaki) in 10% FBS/high glucose DMEM. Recombinant human uPAR protein (R&D Systems), recombinant BDNF (Peprotech), or recombinant IGF-BP2 (Peprotech) was added to the culture at different concentrations (2.5, 5.0, 10, 20, or 50 ng/ml).
6
Isolation of Primary Spinal Cord Neurons
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Primary spinal cord neurons were prepared from Sprague–Dawley (SD) rat embryos (Daehan Bio Link, Chungju, Korea) at the age of 15 embryonic days. Briefly, embryos were promptly separated via cesarean section from pregnant rat and placed in petri dishes containing cold Leibovitz's L-15 medium (Gibco-BRL, Grand Island, NY, USA). Embryonic body was positioned with the abdomen facing up, and the spinal cord was carefully isolated by removing the immature spines. Under magnification, dorsal root ganglia (DRG) and meninges were removed from the isolated spinal cord by using fine forceps. The spinal cords were rinsed once in L-15 medium and were then enzymatically digested using the Neural Tissue Dissociation Kit and the gentleMACS™ Dissociator (both Miltenyi Biotec, Bergisch Gladbach, Germany) for 20 min at 37°C. After dissociation, supernatants were discarded after centrifugation at 2,000 rpm for 3 min. The cell pellet was resuspended in 1 mL neurobasal medium supplemented with B27, GlutaMAX, 1% penicillin/streptomycin (all Gibco-BRL), and 10 ng/mL recombinant BDNF (PeproTech, Rocky Hill, CT, USA). Single cells were then seeded onto coated plates with 20 mg/mL poly-D-lysine overnight and with 10 mg/mL laminin (both Gibco-BRL) for 2 h at 4°C.
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