A mixed primary culture of hypothalamic neurons and glia were prepared from Sprague-Dawley rats pups at P0 and maintained in culture at 37°C and 5% CO2 as previously described (Loktev and Jackson, 2013 (link)) with slight modifications. Briefly, the hypothalamic region of the brain was dissected in ice cold HBSS (HyClone Cat# SH30268.01) containing penicillin-streptomycin solution (MP Biomedicals Cat# 0916700) and sodium pyruvate (GIBCO Cat# 11360070), and was subjected to enzymatic desegregation for 20 min at 37°C in papain buffer containing 20 units/ml papain (Worthington Cat# 3126) and 250 units/ml DNaseI (Worthington Cat# 2006). The tissue was then mechanically disaggregated into isolated cells in a Neurobasal medium (GIBCO Cat# A1371201) with 5% (v/v) horse serum (GIBCO Cat# 26050088), Glutamax-I (GIBCO Cat# 35050–061), B27 supplement (GIBCO Cat# A3582801), and 250 units/ml DNaseI. The cells (1.0 × 106 cells/cm2) were plated in wells of 6-well plates that were coated with 0.1 mg/mL poly-D-lysine (Sigma-Aldrich Cat# P7280). After 2 h, medium was replaced with Neurobasal medium supplemented with B27, Glutamax-I, and penicillin-streptomycin solution. The medium was replaced every 3 days, and the cells were maintained in culture for 5–7 days.
Penicillin streptomycin solution
Manufactured by MP Biomedicals
Penicillin-streptomycin solution is a sterile, liquid solution containing penicillin and streptomycin antibiotics. It is commonly used as a supplement in cell culture media to prevent bacterial contamination.
Automatically generated - may contain errors
Lab products found in correlation
Sodium pyruvate, by Thermo Fisher Scientific (3 mentions)
Horse serum, by Thermo Fisher Scientific (2 mentions)
Glutamax 1, by Thermo Fisher Scientific (2 mentions)
Neurobasal medium, by Thermo Fisher Scientific (2 mentions)
Poly d lysine, by Merck Group (2 mentions)
Dnase 1, by Merck Group (2 mentions)
Papain, by Worthington (2 mentions)
Dnase 1, by Worthington (2 mentions)
Human basic fibroblast growth factor, by Merck Group (1 mentions)
Ciliary neurotrophic factor, by Thermo Fisher Scientific (1 mentions)
Forskolin, by Merck Group (1 mentions)
Platelet derived growth factor aa, by Thermo Fisher Scientific (1 mentions)
B27 supplement, by Thermo Fisher Scientific (1 mentions)
Platelet derived growth factor aa, by Merck Group (1 mentions)
Trace elements b, by Corning (1 mentions)
N2 supplement, by Thermo Fisher Scientific (1 mentions)
D biotin, by Merck Group (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
N acetylcysteine, by Merck Group (1 mentions)
Insulin, by Merck Group (1 mentions)
3 protocols using penicillin streptomycin solution
1
Culturing Hypothalamic Neuron-Glia Primary Cells
2
Isolation and Culture of Mouse Oligodendrocyte Progenitor Cells
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Mouse OPCs were isolated from the cortices of pups at postnatal days 3–8 as described previously.[30 ] Briefly, cortical tissues were dispersed into single cells and the cell suspension was then subjected to immunopanning with antibodies against GalC and O4 sequentially. The enriched Galc‐negative O4‐positive OPCs were plated into poly‐D‐lysine coated dishes and cultured with Mouse OPC growth medium (DMEM/F‐12 (GIBCO, Cat# 11330‐032) supplemented with 1% N2 supplement (GIBCO, Cat# A1370701), 2% B27 supplement (GIBCO Cat# A3582801), penicillin–streptomycin solution (MP Biomedicals, Cat# 0916700), 1% sodium pyruvate (GIBCO, Cat# 11360070), 1% L‐glutamine (Hyclone, Cat# SH30034), 10 ng mL−1 platelet‐derived growth factor‐aa (Peprotech, Cat#100‐13A), 10 ng mL−1 ciliary neurotrophic factor (Peprotech, Cat# 450‐13), 20 ng mL−1 human basic fibroblast growth factor (Sino Biological, Cat# 10014HNAE), 0.5 mg mL−1 insulin (Sigma, Cat# 91077), 5 mg mL−1N‐acetyl cysteine (Sigma, Cat# A8199), 10 ng mL−1 D‐biotin (Sigma, Cat# B4639), 5 mm forskolin (Sigma, Cat# F3917), and 0.1% Trace Elements B (Corning, Cat# 25‐022‐CI)). OPC differentiation medium contained the same components as growth medium except human basic fibroblast growth factor and platelet‐derived growth factor‐aa, but supplemented with 40 ng mL−1 triiodo‐thyronine (Sigma, Cat# T2877).
3
Culturing Hypothalamic Neuron-Glia Primary Cells
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A mixed primary culture of hypothalamic neurons and glia were prepared from Sprague-Dawley rats pups at P0 and maintained in culture at 37°C and 5% CO2 as previously described (Loktev and Jackson, 2013 (link)) with slight modifications. Briefly, the hypothalamic region of the brain was dissected in ice cold HBSS (HyClone Cat# SH30268.01) containing penicillin-streptomycin solution (MP Biomedicals Cat# 0916700) and sodium pyruvate (GIBCO Cat# 11360070), and was subjected to enzymatic desegregation for 20 min at 37°C in papain buffer containing 20 units/ml papain (Worthington Cat# 3126) and 250 units/ml DNaseI (Worthington Cat# 2006). The tissue was then mechanically disaggregated into isolated cells in a Neurobasal medium (GIBCO Cat# A1371201) with 5% (v/v) horse serum (GIBCO Cat# 26050088), Glutamax-I (GIBCO Cat# 35050–061), B27 supplement (GIBCO Cat# A3582801), and 250 units/ml DNaseI. The cells (1.0 × 106 cells/cm2) were plated in wells of 6-well plates that were coated with 0.1 mg/mL poly-D-lysine (Sigma-Aldrich Cat# P7280). After 2 h, medium was replaced with Neurobasal medium supplemented with B27, Glutamax-I, and penicillin-streptomycin solution. The medium was replaced every 3 days, and the cells were maintained in culture for 5–7 days.
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