The cells were grown on 35 mm dishes (Nunc) to a confluency of about 50%. The cells in each dish were transfected with 4 µg of wild-type rCav1.3 or mutant rCav1.3 R930H cDNA in pCMV6b, 1 µg α2δ1 (Supplementary File: Figure S1 ) in pCDNA3.1, 1 µg β2b in pCDNA3.1 and 0.2 µg of pEGFP vector using Jetprime (peqlab, Erlangen, Germany), according to the instructions of the manufacturer. After 48 h, CHO cells were recorded in whole cell configuration at room temperature (22 °C) with an EPC-10 amplifier (HEKA). All recordings were digitized at 10 kHz, low-pass filtered at 2 or 5 kHz, analyzed with PulseFIT software (HEKA) and compensated for 60–70% of the series resistance. The pipettes had a tip resistance of 2.5–4.0 MΩ when filled with a solution containing (in mM): Cs-methane sulfonate 120, CaCl2 5, MgCl2 2, EGTA 10, MgATP 2 and HEPES 10 (pH 7.4 CsOH), yielding a [Ca2+i] of about 110 nM (calculated with WinMAxc). The cells were bathed in a solution containing (in mM): NMDG 130, CaCl2 15, KCl 5 and HEPES 10 (pH 7.4, HCl).
Jetprime
Manufactured by Avantor
Sourced in Germany, United States, United Kingdom
JetPRIME is a versatile transfection reagent used for the efficient delivery of DNA, RNA, and other molecules into a wide range of cell lines. It is a cationic lipid-based formulation designed to facilitate the uptake of nucleic acids and other molecules into the target cells.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin solution, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Mg132, by Merck Group (2 mentions)
Lyovec 2 3 cgamp 3 3 cgamp dmxaa c di gmp, by InvivoGen (2 mentions)
Ripa buffer 9806s, by Cell Signaling Technology (2 mentions)
Lactacystin 70890, by Cayman Chemical (2 mentions)
Celltiter glo, by Promega (2 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (2 mentions)
Poly da dt p0883, by Merck Group (2 mentions)
1 10 phenanthroline, by LifeSensors (2 mentions)
Lps eb, by InvivoGen (2 mentions)
Celltiter blue, by Promega (2 mentions)
Fsl 1, by InvivoGen (2 mentions)
35 mm dishes, by Thermo Fisher Scientific (1 mentions)
Nih3t3 cells, by American Type Culture Collection (1 mentions)
Sodium pyruvate, by Merck Group (1 mentions)
Fugene hd, by Promega (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
L glutamine, by Merck Group (1 mentions)
28 protocols using jetprime
1
Patch-Clamp Analysis of Cav1.3 Calcium Channels
2
Transient Transfection of NIH 3T3 and MEF Cells
3
Cav1.3 Channel Expression in HEK293 Cells
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HEK293 cells were cultured in 25 cm2 flasks at 37°C and 5% CO2 in DMEM (Thermo Fisher Scientific) supplemented with 10% FCS and 1% penicillin/streptomycin solution (Thermo Fisher Scientific). At a confluency of 60–70%, cells were transiently transfected with cDNA encoding Cav1.3 α1-subunits (human, 6 µg/flask) together with auxiliary β2b (human, 3 µg/flask) and α2δ1-subunits (human, 3 µg/flask), EGFP (0.5 µg/flask), and empty vector (pcDNA3.1(+), 3 µg/flask) or VAPB (human, 3 µg/flask) using JetPrime (Peqlab). Cells were subsequently kept at 30°C and 5% CO2. Forty-eight hours after transfection, cells were detached by using 0.05% trypsin and transferred to 35-mm Petri dishes for electrophysiological recordings. Whole-cell patch-clamp recordings were performed at room temperature with an EPC10 amplifier (Heka Electronik) using electrodes pulled from borosilicate capillaries with a resistance of 2–5 MΩ. Series resistance was compensated by 50%. The extracellular solution contained 110 mM NaCl, 20 mM CsCl, 10 mM BaCl2, 10 mM HEPES, 1 mM MgCl2, and 10 mM glucose (pH 7.4). The pipette internal solution contained 130 mM KCl, 10 mM NaCl, 0.5 mM MgCl2, 1 mM EGTA, and 5 mM HEPES (pH 7.3). I–V relationships were obtained by applying a 300 ms long square pulse protocol to various test potentials starting from a holding potential of −80 mV.
4
Cell Culture and Transient Transfection
5
Splicing Analysis of CDKL5 Exon 14
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DNA fragments of wild-type and mutant CDKL5 exon 14 with flanking intron sequence were synthesized (IDT, Coralville, IA) and cloned into the exon trap vector pET01 (MoBiTec, Göttingen, Germany). Minigenes were transfected into HEK293T cells using jetPRIME (PEQLAB, Erlangen, Germany) according to the manufacturer's instructions. After 24 hours, total RNA was extracted using the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany). Total RNA was generated, and reverse transcription PCR (RT-PCR) experiments were performed as described previously.17 (link) Further details of these experiments are described in e-Methods.
6
Generation of Stable Overexpressing Cell Lines
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To produce stably overexpressing cell lines, lentiviral particles were used which were generated by JetPRIME® (PEQLAB Biotechnologie GmbH, Erlangen, Germany) transfection of HEK293T with the lentiviral vectors psPAX2 (Addgene#12260), pMD2.G (Addgene#12259), and cloned vectors as previously described [23 (link)]. Transduced J774A.1 cells were sorted for green positive cells using BD FACSAria™ III cell sorter (BD Biosciences, Heidelberg, Germany).
7
Overexpression and knockdown of key proteins
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HEK293 or HeLa cells were seeded at approximately 30%–40% confluency and transfected 24 hr after plating. Transfection was done using jetPRIME (Peqlab) transfection reagent. After incubation over-night, cell medium was removed and fresh medium was added to the cells. 48 hr after transfection cells were lysed either for protein extraction or RNA purification. For expression of wild-type INSR in HEK293 cells pRT3-INSR-WT (Addgene) was used as template for cloning full-length cDNA of human INSR into pCMV-tag2b vector (Clonetech) (Table S3 ). The K1047R mutation was introduced into pCMV-tag2b-INSR by site-directed mutagenesis (Table S3 ). For overexpression of CHIP pCMV-tag2b-CHIP vector was used (Arndt et al., 2005 (link)). Expression of poly-Q proteins was induced by transient transfection with plasmids pEGFP-polyQ-25 and pEGFP-polyQ-103 (Westhoff et al., 2005 (link)). For efficient depletion two different siRNAs were used per gene (FlexiTube siRNAs CHIP 1+5 and FlexiTube siRNAs ATG7 1+3 (QIAGEN)). Allstars negative control siRNA (QIAGEN) was used as control siRNA.
8
Quantification of Nav1.5 Membrane Expression
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For the quantification of Nav1.5 expression at the plasma membrane, three copies of the hemagglutinin (HA) epitope were inserted at amino acid position 304 in the extracellular S5-S6 loop of domain I of Nav1.5. HeLa cells were transfected with the indicated constructs using jetPRIME (Peqlab). After 48 h, cells were fixed with 4% PFA (w/v) (in PBS), washed three times with PBS and blocked with 10% normal goat serum (v/v) (in PBS). Cells were stained with a monoclonal anti-HA primary antibody (HA-probe (F-7), Santa Cruz, dilution 1:100) and washed 4 times for 15 minutes with PBS. As a secondary antibody a horseradish-peroxidase (HRP)-conjugated antibody (goat anti-mouse IgG-HRP, Santa Cruz, dilution 1:5000) was used. After several washing steps with PBS (6 times for 20 minutes), surface expression was measured as relative light units (RLUs) in a luminometer (GloMax 20/20, Promega) using a luminogenic substrate (SuperSignal ELISA Femto, Thermo Scientific).
9
HeLa Cell Cultivation and Transfection
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HeLa cells were cultivated at 37 °C and 5% CO2 in DMEM medium (Invitrogen) supplemented with 10% FCS and 1% Penicillin/Streptomycin solution (Invitrogen). For imaging experiments cells were grown on glass bottom (WillCo) 35 mm petri dishes, respectively. At a confluency of 60–70% cells were transfected with FuGENE6 (Promega) or JetPRIME (Peqlab). For fluorescence imaging experiments, a total amount of 1 μg of cDNA per 35 mm dish was used for transfection.
10
Detecting Wnt3a Protein Expression
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HEK293T cells and mouse L cells were routinely maintained in Dulbecco’s modified Eagle’s medium (Invitrogen, Waltham, MA, USA) containing 10% fetal bovine serum (FBS) (PAA, Freiburg, Germany) and 1% penicillin/streptomycin in 5% CO2 at 37°C. Cells were split one day prior to transient transfection and grown to 60 to 70% confluence. HEK293T cells were transfected using jetPRIME™ (Peqlab, Erlangen, Germany) reagent (Polyplus Transfection, USA) according to the manufacturer’s protocol. To detect Wnt3a proteins in cell lysates, HEK293T cells were transfected with wild-type Wnt3a or its mutant isoforms. The cells were harvested after 48 hr of transfection, washed with ice-cold 1× PBS and disrupted in lysis buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 1% NP-40, 10 mM ethylenediaminetetraacetic acid (EDTA) protease inhibitor cocktail complete (Roche Diagnostics GmbH, Mannheim, Germany). The resulting supernatant was transferred to a fresh tube. Proteins were analyzed by Western blot using anti-Wnt3a antibody. For loading control, the membranes were probed with anti-α-tubulin.
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