The PRV-TJ strain (GenBank accession number: KJ789182.1 ) was isolated from a pig farm with a PR outbreak in Tianjin, China in 2012 and stored at -70°C and propagated in the porcine kidney 15 (PK-15) cell line (Luo et al., 2014 (link)). PK-15 and Vero cells were obtained from China Center for Type Culture Collection (CCTCC, Wuhan, China) and maintained at 37°C with 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) (Thermo-Fisher Scientific, Carlsbad, CA, United States) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, United States). Dorsal root ganglions (DRGs) were isolated from newborn mice and cultured in Neurobasal medium (Gibco) supplemented with 100 ng/ml nerve growth factor 2.5S (Invitrogen), 2% B-27 (Gibco) and 1% penicillin and streptomycin with 2 mM glutamine (Invitrogen). The Animal Ethics Committee approval number is Heilongjiang-SYXK-2006-032. We conducted all the experiments in Biosafety Level II laboratory following strict biosecurity measures according to instructions of Harbin Veterinary Research Institute.
Nerve growth factor 2.5s
Manufactured by Thermo Fisher Scientific
Nerve growth factor 2.5S is a protein produced by Thermo Fisher Scientific. It is a well-characterized neurotrophic factor that promotes the survival and differentiation of certain neuronal populations.
Automatically generated - may contain errors
Lab products found in correlation
Neurobasal medium, by Thermo Fisher Scientific (4 mentions)
Penicillin and streptomycin, by Thermo Fisher Scientific (4 mentions)
Glutamine, by Thermo Fisher Scientific (4 mentions)
Optical plastic dishes, by Ibidi (2 mentions)
Poly dl ornithine, by Merck Group (2 mentions)
Natural mouse laminin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Cytosine d arabinofuranoside, by Merck Group (1 mentions)
Poly dl ornithine hydrobromide, by Merck Group (1 mentions)
Collagenase dispase, by Roche (1 mentions)
Plastic tissue culture dishes, by BD (1 mentions)
Cytosine d arabinofuranoside ara c, by Merck Group (1 mentions)
4 protocols using nerve growth factor 2.5s
1
Isolation and Cultivation of Porcine Parvovirus
2
Compartmented Culture of Rat SCG and DRG Neurons
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Superior cervical ganglia (SCG) and dorsal root ganglia (DRG) were isolated from E16–17 Sprague-Dawley rat embryos (Hilltop Labs, Inc., Scottsdale, PA) and neurons were cultured in tri-chambers as described (Ch’ng and Enquist, 2005 ). All animal work was performed in accordance with the Princeton Institutional Animal Care and Use Committee (protocols 1947–13 and 1851–14). Multiwell dishes (Falcon), 35-mm plastic tissue culture dishes (Falcon) or optical plastic dishes (Ibidi) were coated with 500 μg/ml of poly-DL-ornithine (Sigma Aldrich) and 10 μg/ml of natural mouse laminin (Life Technologies). To prepare compartmented neuronal cultures, two sets of evenly spaced parallel grooves were etched on the dishes before a silicone grease-coated tri-chamber (Tyler Research) was placed. SCGs were trypsinized and triturated before plating in the Soma (S) compartment. Neurons were maintained in neurobasal medium (Gibco) supplemented with 100 ng/ml nerve growth factor 2.5S (Invitrogen), 2% B27 (Gibco) and 1% penicillin and streptomycin with 2 mM glutamine (Life Technologies). Two days after plating, 1 mM cytosine-D-arabinofuranoside (Sigma-Aldrich) was added to eliminate non-neuronal dividing cells. Neurons were cultured for 14–21 days.
3
Microfluidic Isolation of Axonal Transport
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Dorsal root ganglia (DRG) from the spine of new-born BALB/c mice less than 3 days were collected, washed, and digested by Collagenase/Dispase (Roche) at 37°C in the CO2 incubator for 45–60 min. The neurons were separated from each other sufficiently, filtered, washed, and cultured in Neurobasal Medium (Gibco) supplemented with 100 ng/ml nerve growth factor 2.5S (Invitrogen), 2% B27 (Gibco), and 1% penicillin and streptomycin with 2 mM glutamine (Invitrogen). Before the neuron was planted in one side of the microfluidic chamber, the coverslips were treated with Poly-DL-ornithine hydrobromide (Sigma) for one night and laminin (invtrogen) for at least 6 h and washed with Hanks’ balanced salt solution (HBSS) buffer two times, dried completely, then covered with microfluidic device, neurons were added in one well of microfluidic device, flowed into the chamber connected with two adjacent wells, after 3 days culturing, the axons grow to another chamber along the chamber microgrooves (see illustration). Infection was performed by replacing the Neurobasal Medium in the distal wells and changed with 107 TCID50 rPRV SC-UL36-EGFP. Time-lapse imaging was achieved by automated sequential capture.
4
Culturing Embryonic Rat Superior Cervical Ganglia
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Superior cervical ganglia (SCGs) were isolated from embryonic day 17 Sprague-Dawley rat embryos (Hilltop Labs). Primary neurons were cultured in tri-chamber dishes as previously described [48 ,49 (link)]. Multi-well or 35-mm plastic tissue culture dishes (Falcon) or optical plastic dishes (Ibidi) were coated with 500 μg/ml of poly-DL-ornithine (Sigma Aldrich) and 10 μg/ml of natural mouse laminin (Invitrogen). After coating, two sets of grooves were etched on the dishes. A silicone grease-coated tri-chamber (Tyler Research) was placed on top of a drop of 1% methylcellulose (in neuronal medium) covering the groves. Ganglia were trypsinized and triturated, and approximately 2/3 of an SCG was plated in the S compartment. Neurons were maintained in complete neuronal medium: Neurobasal medium (Gibco) supplemented with 100 ng/ml nerve growth factor 2.5S (Invitrogen), 2% B27 (Gibco) and 1% penicillin and streptomycin with 2 mM glutamine (Invitrogen). 2 to 3 days after seeding, 1 mM cytosine-D-arabinofuranoside (AraC; Sigma-Aldrich) was added for at least 2 days to eliminate non-neuronal cells. Neurons were cultured for 14–21 days prior to experiments.
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