We prepared primary chromaffin cell culture as described previously65 (link). In brief, fresh adult (21 – 27 months old) bovine adrenal glands (local abattoir), were immersed in pre-chilled 1x Lock’s buffer on ice containing: NaCl, 145 mM; KCl, 5.4 mM; Na2HPO4, 2.2 mM; NaH2PO4, 0.9 mM; glucose, 5.6 mM; HEPES, 10 mM; pH 7.3 adjusted with NaOH. Glands were perfused with 1x Lock’s buffer, then infused with Lock’s buffer containing collagenase P (1.5 mg/ml, Roche), trypsin inhibitor (0.325 mg/ml, Sigma) and bovine serum albumin (5 mg/ml, Sigma), and incubated at 37°C for 20 min. The digested medulla was minced in Lock’s buffer, and filtered through a nylon mesh. The filtrate was centrifuged (39 xg, 4 min), re-suspended in Lock’s buffer and re-centrifuged until the supernatant was clear. Final cell pellet was re-suspended in pre-warmed DMEM low-glucose medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and plated onto poly-L-lysine (0.005 % w/v, Sigma) and laminin (4 μg/ml, Sigma) coated glass coverslips. The cells were incubated at 37°C with 8% CO2 and used within 1 week. Before plating, some cells were transfected by electroporation (2 μg plasmid DNA containing NPY-EGFP or VAMP2-EGFP) using Basic Neuron Nucleofector Kit (Lonza, Program O-005).
Basic neuron nucleofector kit
Manufactured by Lonza
Sourced in Switzerland
The Basic Neuron Nucleofector Kit is a lab equipment product designed for the transfection of primary neurons. It provides the necessary components for the efficient delivery of nucleic acids into neuronal cells using the Nucleofector technology.
Automatically generated - may contain errors
Lab products found in correlation
Dmem low glucose medium, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Poly l lysine (pll), by Merck Group (2 mentions)
Laminin, by Merck Group (2 mentions)
Trypsin inhibitor, by Merck Group (2 mentions)
Collagenase p, by Roche (2 mentions)
Nucleofector 2 device, by Lonza (1 mentions)
3 protocols using basic neuron nucleofector kit
1
Preparation of Primary Bovine Chromaffin Cells
2
Primary Schwann Cell Precursor Isolation
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Primary Schwann cell precursors or neurons were prepared from dorsal root ganglia (DRGs) of male or female Sprague-Dawley rats (Sankyo Laboratories) on embryonic day 14 and cultured in culture medium at 37 °C, as previously described [19] , [20] , [21] (link), [22] (link). The siRNAs and/or plasmids were transfected into primary Schwann cell precursors using Nucleofector II device (Lonza, Basel, Switzerland) with Basic Neuron Nucleofector kit (Lonza; a Nucleofector II program A33 protocol). Culture medium was replaced 24 h posttransfection. To confirm cell viability under these experimental conditions, cells were stained with 0.4% trypan blue. Stained attached cells routinely accounted for less than 5% of all cells 48 h posttransfection.
3
Preparation of Primary Bovine Chromaffin Cells
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+ Expand
We prepared primary chromaffin cell culture as described previously65 (link). In brief, fresh adult (21 – 27 months old) bovine adrenal glands (local abattoir), were immersed in pre-chilled 1x Lock’s buffer on ice containing: NaCl, 145 mM; KCl, 5.4 mM; Na2HPO4, 2.2 mM; NaH2PO4, 0.9 mM; glucose, 5.6 mM; HEPES, 10 mM; pH 7.3 adjusted with NaOH. Glands were perfused with 1x Lock’s buffer, then infused with Lock’s buffer containing collagenase P (1.5 mg/ml, Roche), trypsin inhibitor (0.325 mg/ml, Sigma) and bovine serum albumin (5 mg/ml, Sigma), and incubated at 37°C for 20 min. The digested medulla was minced in Lock’s buffer, and filtered through a nylon mesh. The filtrate was centrifuged (39 xg, 4 min), re-suspended in Lock’s buffer and re-centrifuged until the supernatant was clear. Final cell pellet was re-suspended in pre-warmed DMEM low-glucose medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and plated onto poly-L-lysine (0.005 % w/v, Sigma) and laminin (4 μg/ml, Sigma) coated glass coverslips. The cells were incubated at 37°C with 8% CO2 and used within 1 week. Before plating, some cells were transfected by electroporation (2 μg plasmid DNA containing NPY-EGFP or VAMP2-EGFP) using Basic Neuron Nucleofector Kit (Lonza, Program O-005).
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