Neuron cultures were obtained from C57BL/6 male mice of 8 weeks. All experimental procedures were carried out in compliance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines and in accordance with National Institute of Health guidelines for animal care and use of Laboratory animals (DL 2016, Italian Ministry of Health approval protocol 919/2015-PR). A complete dorsal root ganglia (DRG) pool was collected in F12 medium (Euroclone, Italy) from each mouse. DRG were digested for 1 h with 12.5 mg/ml collagenase (Sigma Aldrich, USA) and 10 mg/ml DNase (Sigma Aldrich, USA) and then mechanically triturated. A BSA gradient (30% BSA, 70% F12 medium) was used to isolate neurons that were then resuspended in BS medium (F12 medium supplemented with 1% N2 supplement 100X (Life Technologies, UK), 1% BSA (Sigma Aldrich, USA), 1% Penicillin and Streptomycin 100X (Euroclone, Italy), 1% L-glutamine 100X (Euroclone, Italy) and seeded in a single drop on poly-l -lysine (Sigma Aldrich, USA) coated dishes. After 24 h, neurons cultures were treated with 10−5 M 2′-Deoxy-5-fluorouridine (FuDR) (Sigma, USA) to remove satellite cells.
F12 medium
Manufactured by Euroclone
Sourced in Italy
F12 medium is a cell culture medium formulation commonly used for the growth and maintenance of various cell lines. It provides the necessary nutrients and components required for cell proliferation and survival in in vitro cell culture applications.
Automatically generated - may contain errors
Lab products found in correlation
Collagenase, by Merck Group (1 mentions)
2 deoxy 5 fluorouridine fudr, by Merck Group (1 mentions)
N 2 supplement 100x, by Thermo Fisher Scientific (1 mentions)
L glutamine 100x, by Euroclone (1 mentions)
F12 medium, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Dnase, by Merck Group (1 mentions)
Gentamicin sulfate, by Euroclone (1 mentions)
Sodium pyruvate, by Euroclone (1 mentions)
Fetal bovine serum (fbs), by Euroclone (1 mentions)
L glutamine, by Euroclone (1 mentions)
2 protocols using f12 medium
1
Isolation of Murine Dorsal Root Ganglia Neurons
2
Characterizing BMP6 Peptide Binding in CHO Cells
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Wild-type Chinese Hamster Ovary (CHO-K1 ATCC® CCL-61) and mutant CHO-745 (pgsA-745 ATCC® CRL-2242 [16, 17] ) cells were maintained at 37 • C and 5% CO 2 , in F12 medium (Euroclone) supplemented with 10% Fetal Bovine Serum (FBS, Euroclone), 40 μg/mL gentamicin sulfate (Euroclone), 2 mM L-glutamine (Euroclone) and 1 mM sodium pyruvate (Euroclone). Wild type and mutant CHO cells were seeded in a TC 96-well plate (Sarstedt) with a cellular density of 80.000 cells/well. Once the monolayers formed, the medium was removed, and cells were washed gently with PBS, fixed with 3% glutaraldehyde, incubating for 2 h at 4 • C, while the reaction was stopped adding glycine at a final concentration of 0.1 M. After PBS washing, the non-specific binding sites were saturated with PBS + 3% BSA. Synthetic BMP6 peptides (5, 25, 50, 250 μM in PBS + 3% BSA) were spotted onto cell monolayers and incubated 3 h at 37 • C. Unbound peptides were removed by washing with PBS, while cell-bound peptides were detected as previously described, taking advantage of their biotin tag.
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