ASOs (Supplementary Table S6 ) were synthesized by IDT technologies. ASOs (20 nM) were transfected into RAW264.7 cells with the X-tremeGENE HP DNA (Roche) according to the manufacturer’s protocol. To maximize knockdown efficiency, ASO transfection was repeated 24 hours after the initial transfection. Cells were then treated with LPS for 2 hours.
X tremegene hp dna
Manufactured by Roche
Sourced in United States, Switzerland, Germany
X-tremeGENE HP DNA is a transfection reagent for the efficient delivery of DNA into a wide range of cell types. It facilitates the uptake of DNA into cells, enabling the expression of the introduced genetic material.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
X tremegene sirna transfection reagent, by Roche (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Hek293, by American Type Culture Collection (2 mentions)
Rapamycin, by Selleck Chemicals (2 mentions)
X tremegene sirna, by Roche (2 mentions)
Mk 2206, by Selleck Chemicals (2 mentions)
Pgl3 basic vector, by Promega (2 mentions)
Quikchange 2 xl kit, by Agilent Technologies (2 mentions)
Dual luciferase reporter assay system, by Promega (2 mentions)
Prl sv40 renilla vector, by Promega (2 mentions)
Victor x3 plate reader, by PerkinElmer (2 mentions)
293t cell, by American Type Culture Collection (1 mentions)
Bright glo, by Promega (1 mentions)
Bglii restriction enzyme, by New England Biolabs (1 mentions)
Fetal bovine serum (fbs), by Harvard Bioscience (1 mentions)
Penicillin, by Harvard Bioscience (1 mentions)
Streptomycin, by Harvard Bioscience (1 mentions)
Taqman assay, by Thermo Fisher Scientific (1 mentions)
41 protocols using x tremegene hp dna
1
RAW264.7 Cells ASO Knockdown
2
HIV Envelope Pseudotyped Virus Neutralization Assay
3
Generation and Maintenance of HEK 293 Cells
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HEK 293 cells (DSMZ—German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany; RRID:CVCL_0045) were maintained in DMEM supplemented with FBS (Biochrom; Berlin Germany), penicillin (100 U·ml−1; Biochrom), and streptomycin (100 μg·ml−1; Biochrom), with or without geneticin (G418, 100 μg·ml−1; Biochrom), in 5% CO2 at 37°C. Cells were passaged 1:3–1:20 every second to third day from P8 to P28 depending on confluence; 24 hr after seeding, confluent HEK WT cells (70–90%) were incubated with 1 μg per 200‐μl transfection mix of each plasmid containing the different cDNAs using X‐treme GENE HP DNA (Roche, Mannheim, Germany) reagent following the supplier's recommendations. For stable transfection, pcDNA™3.1 carrying the cDNA of MOR‐H2976.52A was linearized with restriction enzyme BglII (NEB, Frankfurt am Main, Germany). After 48 hr, the medium containing the transfection reagent was removed and geneticin (G418; 100 μg·ml−1) was added with standard culture medium for selection of successfully transfected cells. Membrane expression of the transfected receptors was qualitatively verified via immunocytochemistry (data not shown). Stably transfected, polyclonal cell lines were cultured for a maximum of 23 passages.
4
Transfection Efficiency Evaluation
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IC-21 and HT-29 cell lines were maintained in RPMI1640 media supplemented with 20% and 10% fetal bovine sera, respectively. DNA (137-bp fragment or pGFP) was delivered into the cells using FND-PEI (25 µg per ml FNDs), PEI (3.3 µg ml -1 , equivalent to the amount that covers FND surfaces) or the commercial transfection reagent X-tremeGENE HP DNA (Roche; 3 : 1 ratio, here, the manufacturers protocol has been followed). After 18 h incubation, we detected the level of transfected 137-bp fragment by real-time PCR with a TaqMan® probe using a commercial set of internal primers and probes for mouse beta-actin (TaqMan® Assay, Life Technologies). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene amplification was used as a reference, and quantitative levels were determined with Bio-Rad iQ5 2.0 software. All samples were performed in biological and technical triplicates. The 137-bp DNA fragment amplified and/or FAMlabeled was used in confocal analyses, PL measurements and toxicity assays.
5
Nanoparticle-Mediated Oligonucleotide Delivery
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The equivalent of 10 5 cells IC-21 maintained in RPMI1640 media supplemented with 20% fetal bovine sera was seeded on a 12-well plate (In Vitro Scientific, USA). The AlexaFluor 488-modified oligonucleotide (0.25 µl, 1 mM solution, see Preparation of DNA) was incubated with a 12.5 µl FND-PEI complex (1 mg per ml of FND-PEI, sonicated for 30 minutes) for 60 minutes at room temperature. The FND-PEI-DNA complex was isolated by centrifugation at 14 000g for 15 min, and the pellet was resuspended in an appropriate amount of DNAse-free water (Qiagen) to obtain a stock solution of FND-PEI-DNA (1 mg ml -1 ). IC-21 cells were incubated with the FND-PEI-DNA complex (final concentration 25 µg ml -1 ) for 30, 60, and 120 min. In parallel cell samples, an equal amount of the oligonucleotide was delivered into the cells using the commercial transfection reagent X-tremeGENE HP DNA (Roche; 3 : 1 ratio; the manufacturers protocol has been followed). At the end of the incubation period, cells were harvested, washed, and resuspended in PBS. Before the flow cytometry analysis, dead cells were stained with Hoechst 33258 (staining dead cells) for 5 minutes and the samples were measured with a flow cytometer BD LSR II and analyzed with a FlowJo 7.2.2 software.
6
Visualizing ER Calcium Dynamics in HeLa Cells
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Before Ca2+ imaging, 30,000 cells were plated in NuncTM Labtek® chambered coverglass. For subcellular ER Ca2+ imaging, HeLa cells were transfected with R-CEPIA1er probe, 2 days prior to imaging using X-tremeGENE HP DNA (Roche). Before the experiment, cells were incubated with a Ca2 + -free Balanced Salt Solution (BSS) (121 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl2, 6 mM NaHCO3, 5.5 mM D-glucose, 25 mM HEPES, pH 7.3). After 10 s of measurement, either 100 μM histamine or 10 μM of Thapsigargin in BBS. Six samples were analyzed per experiment. At least three independent experiments were carried out.
For calcium refilling assays, cells were loaded with 5 μM of FluoForteTM Ca2+ probe (Enzo) in BBS during 30 min at 37 °C. After injection of 10 µM of thapsigargin, 2 mM of Ca2+ was added to the mix.
For Calcium pool measurement, cells were transfected with GEM CEPIA1er, 2 days prior to imaging, and incubated in BBS with 2 mM of Ca2+. Time-lapse fluorescence values were collected using a Zeiss LSM 780 confocal microscope. The images were captured at a rate of one frame every 2 s using a ×40 objective. All images were analyzed by ImageJ software.
For calcium refilling assays, cells were loaded with 5 μM of FluoForteTM Ca2+ probe (Enzo) in BBS during 30 min at 37 °C. After injection of 10 µM of thapsigargin, 2 mM of Ca2+ was added to the mix.
For Calcium pool measurement, cells were transfected with GEM CEPIA1er, 2 days prior to imaging, and incubated in BBS with 2 mM of Ca2+. Time-lapse fluorescence values were collected using a Zeiss LSM 780 confocal microscope. The images were captured at a rate of one frame every 2 s using a ×40 objective. All images were analyzed by ImageJ software.
7
Transient Transfection of Connexin Mutants
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Connexin-deficient mouse neuroblastoma (N2A) and human cervical carcinoma (HeLa) cells (American Type Culture Collection, Manassas, VA, USA) were cultured at 37 °C with 5% CO2. Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Cat# 10313-021, Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum, 1% penicillin, 1% streptomycin, 4.5 g/L d -(+)-glucose, 584 mg/L l -glutamine, and 110 mg/L sodium pyruvate. Twenty-four hours before cell transfection, N2A or HeLa cells were replated into a 35-mm dish at 60% confluency. Transfection was performed the next day by adding 0.8–1 μg of DNA with 2 μL of the transfection reagent X-tremeGENE HP DNA (Roche Applied Sciences, Indianapolis, IN, USA). To assess the effect of Cx40 mutants on wildtype Cx43, N2A cells were transfected in a 1:1 ratio of Cx40 mutants-IRES-GFP and Cx43-IRES-DsRed. Cell pairs successfully co-expressing both GFP and DsRed were selected for measuring coupling conductance with dual whole-cell patch clamp (see below).
8
Investigating Gene Expression via siRNA and Plasmid Transfection
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Cells cultured on six-well plates were transfected with siRNA or non-specific control siRNA (si-NC) using Lipofectamine 2000 (Invitrogen, Shanghai, China), according to the manufacturer's protocol. The sequences for siRNAs are listed in Supplemental Table S3 . The PTTG3P plasmid was synthesized according to the sequence of RACE (based on the PTTG3P sequence, NR_002734.2, in NCBI) and then subcloned into a pCDNA3.1 vector (Invitrogen, Shanghai, China). An empty pCDNA3.1 vector was used as a control. Plasmid transfection was carried out by using X-tremeGENE™ HP DNA (Roche, Basel, Switzerland). Cells were harvested after 48 h for qRT-PCR and other experiments.
9
PTTG3P Regulation of Luciferase Activity
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The specific DNA fragments containing the wild type or mutant PTTG3P fragment were sub-cloned downstream of the luciferase gene within the pGL3-Baisc luciferase reporter vector (GENECHEM Co., Ltd., Shanghai, China). Human A549 cells (1.0×105) grown in a 24-well plate were co-transfected with 500 ng of either empty vector or E2F1 construct; 500 ng of firefly luciferase reporter comprising wild type or mutant PTTG3P using X-tremeGENE™ HP DNA (Roche, Shanghai, China) was used. Forty-eight hours after transfection, luciferase assay was performed by using the Dual-Luciferase Kit (Promega, USA). The relative firefly luciferase activities were normalized to those of Renilla luciferase. All samples were assayed in triplicate.
10
Co-immunoprecipitation of P4-ATPases and CDC50A
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For co-immunoprecipitation, HEK293 cells were transfected with pEGFP-P4-ATPases and pCDC50A-FLAG using X-tremeGENE HP DNA (Roche). Two days after transfection, the cells were lysed in cold buffer (150 mM NaCl, and 50 mM Tris at pH 7.4 containing 1% Triton X-100 and protease inhibitors). After removal of insoluble fractions by centrifugation, the supernatants were incubated with anti-FLAG M2 affinity gel (Sigma) for 30 min on ice. After washing with 0.1% Triton X-100/TBS, immunoprecipitates were eluted with FLAG peptides (500 μg/mL, Sigma) for 30 min on ice. Resulting supernatants were incubated in SDS sample buffer at 50 °C for 5 min.
For immunoblotting, proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membrane. The membrane was blocked in TBS containing 5% skim milk and 0.1% Tween 20, stained with antibodies and visualized using an enhanced chemiluminescent reagent. Uncropped immunoblots are shown in Supplementary Figure8 .
For immunoblotting, proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membrane. The membrane was blocked in TBS containing 5% skim milk and 0.1% Tween 20, stained with antibodies and visualized using an enhanced chemiluminescent reagent. Uncropped immunoblots are shown in Supplementary Figure
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