SAG siRNA (Silencer select predesigned siRNA) and control (scrambled) siRNA were purchased from Ambion (Carlsbad, CA, USA) and Invitrogen, respectively. siRNA transfection into J774, RAW264.7 or BMDM cells (at 8 × 105 cells per well of a 6-well plate) was conducted using 8 μl X-tremeGENE Transfection Reagent (Roche) with 90 pmol of siRNAs per well. HA-SAG-pcDNA3 and pcDNA3 plasmids were used for SAG overexpression study. For BMDM cells, DNA transfection was conducted with 8 × 105 cells per well of a 6-well plate using 4 μl X-tremeGENE Transfection Reagent (Roche) with 2 μg DNA plasmids. The transfection efficiency for siRNA knockdown was determined by immunodetection of SAG protein and real-time PCR of SAG mRNA (Supplementary Figure S2 ). The transfection efficiency for cDNA overexpression was determined by immunodetection of the HA-tag protein (Supplementary Figure S10 ). At 16 h after transfection, cells were treated with PAMP(s) for different time periods.
X tremegene transfection reagent
Manufactured by Roche
Sourced in United States, Switzerland, Germany, China, Spain
X-tremeGENE is a transfection reagent. It is designed to facilitate the delivery of nucleic acids, such as plasmid DNA, into mammalian cells.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (11 mentions)
Opti mem media, by Thermo Fisher Scientific (7 mentions)
Dual luciferase reporter assay system, by Promega (6 mentions)
Opti mem, by Thermo Fisher Scientific (5 mentions)
Turbofect transfection reagent, by Thermo Fisher Scientific (4 mentions)
Puromycin, by Merck Group (4 mentions)
Mcf 7, by American Type Culture Collection (4 mentions)
Si nc, by RiboBio (4 mentions)
Ripa buffer, by Beyotime (3 mentions)
Mda mb 231, by American Type Culture Collection (3 mentions)
Non targeting sirna, by GenePharma (3 mentions)
Opti mem, by Roche (3 mentions)
Lenti x concentrator, by Takara Bio (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (3 mentions)
Lsm 710, by Zeiss (2 mentions)
Hiperfect transfection reagent, by Qiagen (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions)
Specific human sirnas against p38γ, by GenScript (2 mentions)
196 protocols using x tremegene transfection reagent
1
SAG Modulation in Macrophage Cells
2
Fluorescence-Based Protein-Protein Interactions
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V-PAC assays were performed as described previously with a minor modification by using more potent Venus fluorescent proteins instead of original Yellow Fluorescent proteins [49 (link)]. Briefly, 1 million of HeLa cells were transfected with 0.4μg of plasmid DNA encoding YC-P-TEFb and 1.2μg of plasmid DNA encoding VN-CTD using X-tremeGENE Transfection Reagents (Roche). Twenty-four hours post-transfection, the cells were split into 24-well plates and kept in 5% FCS. At 48 hours post-transfection, the cells were mock-treated or treated with the different compounds as indicated for 60 min. Fluorescence signals were detected by microscopic analysis using Olympus IX70 bright field fluorescent microscope. The fluorescence images were analyzed using Metamorph software and Venus positive cells were manually counted and averaged from three randomly chosen fields of each sample.
3
Immunoprecipitation of HA- and Flag-tagged Proteins
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293T cells were transfected in 6-well dishes by use of X-tremeGENE transfection reagents (Roche Diagnostics) according to the manufacturer’s directions. Approximately 48 h after transfection, cells were washed with PBS and lysed in 400 μl of M-PER (Thermo Scientific) with additional protease inhibitors. After centrifugation at 4°C for 15 min, 20 μl of cell extract was stored as input sample; the remaining protein was incubated with 15 μl anti-HA or anti-Flag–agarose (Thermo Scientific) and rotated at 4°C for 4 h. Samples were washed three times with Tris-buffered saline (TBS) plus 0.05% Tween 20 (TBS-T), and then 25 μl nonreducing sample buffer (2×) was added and the mixture was heated for 5 min. Samples were analyzed by Western blotting using the indicated antibodies.
4
Cell Transfection and MafB Silencing
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Detailed procedures are described in our previous work5 (link). RAW 264.7 and THP-1 cells are transfected using TurboFect transfection reagents (Thermo Fisher Scientific) and the Nucleofector™ kit (Lonza), respectively, according to the manufacturer’s recommendations. For transfection of MafB siRNA (156036, Ambion) (50 nM), X-tremeGENE Transfection Reagents (Roche) was used.
5
Cell Culture and Transfection Protocol
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COS-1 and CHO cells were cultured in DMEM or DMEM/F12 respectively, supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. Transfection was performed using X-tremeGENE Transfection Reagents from Roche following the procedure recommended by the company.
6
Photosensitive Neural Cell Culture
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Neuro2a cells, derived from mouse neuroblastoma cells55 56 (link), were used as a model of the neural cells. Neuro2a cells were transfected with both a photosensitive cation channel protein (ChR257 (link)58 (link)) and a red-fluorescent-protein-based genetically encoded Ca2+ indicator (R-GECO16 (link)) using X-tremeGENE transfection reagents (Roche Applied Science, Germany). These gene expression plasmids were obtained from Addgene (ChR2 #15753, R-GECO1 #32444). The transfected cells were embedded within a collagen gel (Cellmatrix Type I-A, I-P, Nitta Gelatin Inc., Japan) or an extracellular matrix gel (E1270, Sigma-Aldrich, USA) in a manner similar to that previously described37 (link), and the liquid gel was pasted on the sensor, which was placed in a dish (Figs 3 b and 4 a). After gel solidification by warming at 37°C, the sensor was then immersed in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS) and antibiotics, and was cultured at 37 °C and 5% CO2 in an incubator.
7
Culturing and Transfecting Common Cell Lines
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MCF10A (CRL-10317), 293T (CRL-11268), MDCK (CCL-34), Caco2 (HTB-37), A431 (CRL-1555), and HeLa (CCL-2) cell lines were obtained from ATCC. All experiments were performed on cell lines that had been passaged for <3 mo after thawing. 293T, A431, and HeLa cells were cultured in DMEM, Caco2 in RPMI, and MDCK in Eagle’s minimum essential medium (Sigma-Aldrich). Culture media were supplemented with 10% FBS (Gibco), 200 U/ml penicillin, and 200 µg/ml streptomycin (Sigma-Aldrich). MCF10A cells were cultured as described previously (Debnath et al., 2003 (link)). Transient gene expression in MCF10A cells was performed by using X-tremeGENE Transfection Reagents (Roche) and in HeLa cells by using Lipofectamine (Thermo Fisher Scientific).
8
Assessing PPARγ Transcriptional Activity
9
Silencing SDPR-AS in 769-P cells
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Small interfering RNAs (siRNAs) for the target SDPR-AS (SDPR-AS-si) and negative-control siRNAs (NC-si) were obtained from GenePharma (Shanghai, People’s Republic of China), and the sequences are listed in Table 1 . The SDPR-AS high-expressing 769-P cell line was selected for transfection with SDPR-AS-si. Prior to transfection, 12-well plates were seeded with approximately 50% of the cells and cultured for 24 h. Then, siRNA transfections were carried out with X-treme GENE transfection reagents (Roche) according to the manufacturer’s instructions. RNA was isolated after 48 h of transfection and used to perform functional assays.
10
Real-Time Monitoring of Intracellular cAMP
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