Primary cultured mixed glial cells and highly-enriched microglia and astrocytes were cultured directly on poly-D-lysine/laminin coated glass coverslips (Corning Biocoat, German) in 24-well plates at 1–2 × 105 cells/ml with complete medium for 24 h. Cells were washed with PBS and fixed with 4% paraformaldehyde for 20 min on ice. Using a similar protocol as described previously36 (link). For brain histochemistry, mice were sacrificed and brains processed as described previously17 (link).
Poly d lysine laminin coated glass coverslips
Manufactured by Corning
Sourced in United States
Poly-D-lysine/laminin coated glass coverslips are a type of lab equipment. They provide a surface coating that promotes cell adhesion and growth.
Automatically generated - may contain errors
Lab products found in correlation
Glutamax, by Thermo Fisher Scientific (1 mentions)
L glutamic acid, by Merck Group (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Dmem f12 media, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Trypsin inhibitor, by Merck Group (1 mentions)
Collagenase a, by Roche (1 mentions)
Collagenase d, by Roche (1 mentions)
Papain, by Worthington (1 mentions)
4 protocols using poly d lysine laminin coated glass coverslips
1
Primary Glial Cell Culture and Histochemistry
2
Culturing Dissociated Rat Hippocampal Neurons
3
Tetrodotoxin-Resistant Nav1.7 Isoform Expression
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The human adult-long splice Nav1.7 isoform complementary DNA (cDNA)
has been previously described.14 (link) Briefly, the cDNA was cloned into a mammalian expression vector and
converted to a tetrodotoxin-resistant phenotype by Y362S substitution
(hNav1.7R/AL, hereafter referred to as WT). The
hNav1.7R/AL-V810M missense mutation (hereafter
referred to as V810M) was introduced using QuickChange XL site-directed
mutagenesis (Stratagene, San Diego, CA). Human embryonic kidney 293 (HEK293)
cells seeded onto 12 mm poly-D -lysine/laminin coated glass coverslips
(Corning, Corning, Inc., Corning, NY, USA) were cotransfected with either WT or
V810M plasmids (0.8 µg/well) and human β1 and β2 subunits (0.2 µg/well each)
using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). HEK293 cells
were maintained under standard culture conditions (37°C with 5% CO2)
in Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium supplemented with 10%
fetal bovine serum and 1% penicillin/streptomycin (hereafter referred to as DRG
medium).
has been previously described.14 (link) Briefly, the cDNA was cloned into a mammalian expression vector and
converted to a tetrodotoxin-resistant phenotype by Y362S substitution
(hNav1.7R/AL, hereafter referred to as WT). The
hNav1.7R/AL-V810M missense mutation (hereafter
referred to as V810M) was introduced using QuickChange XL site-directed
mutagenesis (Stratagene, San Diego, CA). Human embryonic kidney 293 (HEK293)
cells seeded onto 12 mm poly-
(Corning, Corning, Inc., Corning, NY, USA) were cotransfected with either WT or
V810M plasmids (0.8 µg/well) and human β1 and β2 subunits (0.2 µg/well each)
using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). HEK293 cells
were maintained under standard culture conditions (37°C with 5% CO2)
in Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium supplemented with 10%
fetal bovine serum and 1% penicillin/streptomycin (hereafter referred to as DRG
medium).
4
Isolation and Culture of Rat DRG Neurons
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Adult male Sprague Dawley rats (6-7 weeks old) were used according to the Catholic University of Korea Institutional Animal Care and Use Committee guidelines. L4-L6 DRG neurons were isolated and incubated in a complete saline solution (in mM: 137 NaCl, 5.3 KCl, 1 MgCl2, 25 D-sorbitol, 3 CaCl2, and 10 HEPES at a final pH of 7.2, adjusted with NaOH), as described previously by Rizzo et al.17 (link) and enzymatically digested with collagenase A (1 mg/mL; Roche, Indianapolis, IN) and then with collagenase D (1 mg/mL; Roche) and papain (30 U/mL; Worthington, Lakewood, NJ) for 20 min at 37°C, and then gently centrifuged (900 rpm for 3 min). Pellets were triturated in 1:1 DMEM/F-12 media (Thermo Fisher Scientific, Waltham, MA) with 10% FBS and 100X Penicillin-Streptomycin (Thermo Fisher Scientific) containing bovine serum albumin (1.5 mg/mL; Sigma, S Louis, MO) and trypsin inhibitor (1.5 mg/mL; Sigma). Cells were plated on 12 mm Poly-D-Lysine/Laminin coated glass coverslips (Corning, Corning, NY). The electrophysiological recording was started after 2 hours when DRG cells were stabilized and attached well on coverslips.
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