Primary cultures of embryonic hippocampal neurons were prepared from CD1 mice on embryonic day 17–18. Pregnant animals were sacrificed by cervical dislocation. Embryonic hippocampi were isolated aseptically and hippocampal tissue was digested and triturated according to Czöndör et al.44 (link). Cells were seeded onto poly-l -lysine-laminin (Sigma-Aldrich) -coated glass coverslips in 24-well plates at 6.3–6.8 × 104 or 6-well plates at 4.7–5 × 104 cells/cm2 density and cultivated at 37 °C in 5% CO2 atmosphere. Neurobasal medium (Invitrogen) containing 2% B27 supplement (Invitrogen), 0.5 mM Glutamax (Gibco) and 5% FCS (Invitrogen) was used for plating and for a complete medium change on the first day after plating (DIV1). On the 5th, 9th and 12th day after plating, third of the culture medium was changed to FCS-free fresh medium. To inhibit glial cell division, 10 µM cytosine β-d -arabinofuranoside (Sigma-Adrich) was added to the cultures between DIV4 to 6. To achieve a complete blockade of firing, cell cultures were treated with 1 µM tetrodotoxin (TTX, Tocris) between DIV12–14.
Poly l lysine laminin
Manufactured by Merck Group
Poly-l-lysine-laminin is a compound used for cell culture applications. It consists of the polycationic polymer poly-l-lysine and the extracellular matrix protein laminin. This compound is used to promote cell adhesion and growth on various surfaces.
Automatically generated - may contain errors
Lab products found in correlation
Neurobasal medium, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
B27 supplement, by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Cytosine β d arabinofuranoside, by Merck Group (1 mentions)
Transit 293, by Mirus Bio (1 mentions)
Pegfp n1, by Takara Bio (1 mentions)
Dnase 1, by Merck Group (1 mentions)
35 mm glass bottom dish, by MatTek (1 mentions)
Papain, by Worthington (1 mentions)
Recombinant mouse noggin, by R&D Systems (1 mentions)
Matrigel, by BD (1 mentions)
Tryple, by Thermo Fisher Scientific (1 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (1 mentions)
4 protocols using poly l lysine laminin
1
Embryonic Hippocampal Neuron Culture
2
Culturing and Transfecting Embryonic Hippocampal Cells
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Primary cultures of embryonic hippocampal cells were prepared from CD1, C57Bl/6, or Rin1−/− mice on embryonic day 17/18 according to Czöndör et al. (2009) (link). Cells were seeded onto poly-l -lysine/laminin (Sigma)–coated glass coverslips in 24-well plates or into six-well plates at 1.3 × 105 or 4.5 × 105 cells/well, respectively. For live-cell imaging experiments, cells were seeded into poly-l -lysine/laminin-coated glass-bottom 35-mm Petri dishes (Greiner) with the same cell density. Cells were transfected using Lipofectamine 2000 (Invitrogen) with the following constructs: pEGFP-N1 (Clontech), pEGFP-C2 hRIN1 wt, pEGFP-C2 hRIN1 S351A, and pEGFP-C2 hRIN1 S292A (described in Ziegler et al., 2011 (link)). The plasmids encoding pEGFP-C2-hRIN1 E574A and pEGFP-C2-hRIN1-E574A/S351A were generated by QuikChange site-directed PCR mutagenesis following the manufacturer’s instructions (Stratagene). To generate pEGFP-C2-hRIN1 QM (Y36F, Y121F, Y148F, Y295F), the cDNA encoding hRIN1 QM (generated by PCR using M4-hRIN1 QM as a template) was subcloned into a pEGFP-C2 vector.
HEK293T cells were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum. Cells were transfected using TransIT293 (Mirus Bio, Madison, WI) according to the manufacturer’s instructions with the following constructs: pEGFP-N1, pEGFP-C2 hRIN1 wt, pEGFP-C2 hRIN1 S351A, and pEGFP-C2-hRIN1 E574A.
HEK293T cells were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum. Cells were transfected using TransIT293 (Mirus Bio, Madison, WI) according to the manufacturer’s instructions with the following constructs: pEGFP-N1, pEGFP-C2 hRIN1 wt, pEGFP-C2 hRIN1 S351A, and pEGFP-C2-hRIN1 E574A.
3
Dissociation and culture of primary neurons
4
Neural Differentiation of Human Embryonic Stem Cells
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Neural differentiation from hESC line H1 (WiCell) was performed as described (26 (link)). Briefly, hESCs were split by ethylenediaminetetraacetic acid (EDTA) (Ambion) and cultured in N2B27 (1:1 mix of D-MEM/F12 supplemented with N2 and Neurobasal medium supplemented with B27, all from Gibco) with 100 ng/ml mouse recombinant noggin (R&D Systems), on a matrigel (BD Biosciences) or poly-L-lysine/Laminin (Sigma-Aldrich) coated plate. Subsequently, cells were split using collagenase and cultured in N2B27 and noggin. After about 3–4 weeks, cells were split using TrypLETM (Gibco) and cultured in N2B27, supplemented with 20 ng/ml basic fibroblast growth factor (bFGF) (PeproTech).
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