For each tissue preparation, mice were euthanized via induction of isoflurane briefly followed by decapitation. The spinal column was removed and bisected; lumbar DRG were removed and pooled, and subjected to enzymatic incubations as described [20 (link),57 ,84 ]. DRG tissue was incubated in papain (45 U in HBSS+H, Worthington) for 20 min at 37°C, rinsed, and incubated in collagenase (1.5 mg/mL in HBSS+H, Sigma-Aldrich) for 20 min. DRG were washed again with HBSS+H, then transferred to 1mL of DRG media, which consisted of Neurobasal A medium (Invitrogen) supplemented with 100 U/mL penicillin/streptomycin (Corning), 2 mM GlutaMAX (Life Technologies), 2% B27 (Gibco) and 5% fetal bovine serum (Gibco). DRG were then manually triturated with fire-polished Pasteur pipettes (VWR) and passed through a 40-μm filter (VWR). DRG were then plated onto poly-d-lysine/collagen (Sigma-Aldrich)-coated 12-mm glass coverslips (Thermo Fisher Scientific) and stored at 37°C and 5% CO2 until testing.
Poly d lysine collagen
Manufactured by Merck Group
Poly-d-lysine/collagen is a laboratory product composed of a combination of the synthetic polymer poly-d-lysine and the natural protein collagen. It is typically used as a substrate coating for cell culture applications to promote cell adhesion and growth.
Automatically generated - may contain errors
Lab products found in correlation
40 μm filter, by Avantor (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Pasteur pipette, by Avantor (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Neurobasal a medium, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Corning (2 mentions)
12 mm glass coverslips, by Thermo Fisher Scientific (1 mentions)
Collagenase, by Merck Group (1 mentions)
Hank s buffered salt solution, by Corning (1 mentions)
Hepes, by Merck Group (1 mentions)
Papain, by Worthington (1 mentions)
2 protocols using poly d lysine collagen
1
Isolation and Culture of Mouse Dorsal Root Ganglia
2
Dorsal Root Ganglion Neuron Isolation
Check if the same lab product or an alternative is used in the 5 most similar protocols
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For each tissue preparation, two age- and sex-matched mice were killed by live decapitation, and cervical through lumbar DRG were removed and pooled together. DRG were incubated in papain (45 U, Worthington) for 20 min at 37°C, 5% CO2. DRG were then rinsed and incubated in collagenase (1.5 mg/mL, Sigma-Aldrich) for 20 min. Both enzyme solutions were made up in Ca2+- and Mg2+-free Hanks’ buffered salt solution (Corning) with 10 mm Hepes (Sigma-Aldrich). DRG were manually triturated with fire-polished Pasteur pipettes (VWR) to dissociate neurons, passed through a 40-μm filter (VWR), and plated onto poly-d -lysine/collagen (Sigma-Aldrich)-coated 12-mm glass coverslips (Thermo Fisher Scientific). Neurons were maintained in culture for 2 d in Neurobasal A medium (Invitrogen) supplemented with 100 U/mL penicillin/streptomycin (Corning), 2 mm GlutaMAX (Life Technologies), 2% B27 (Gibco), and 5% fetal bovine serum (Gibco).
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