For transient transfection, the cells were cultured in six-well culture plates prior to transfection. Either siRNA or a control oligonucleotide (A10001, Gene Pharma, Jiang Su, China) was incubated with 9 μl of Lipofectamine RNAiMAX lipid reagent (13778150, Invitrogen, USA) in 150 μl of Opti-MEM medium (31985070, Gibco) for 5 min at RT. A final volume of 250 μl of C3 siRNA or the control oligonucleotide complex was added to the cell culture plates. After 24 h, CSE was added, and the cells were stimulated for another 24 h. Finally, the cells were harvested for subsequent experiments. The primer sequences were: siControl Negative control FAM: 5′-UUCUUCGAACGUGUCACGUTT (forward), 5′-ACGUGACACGUUCGGAGAATT -3′ (reverse); siC3 C3-homo-66: 5′-GUCCCAUGUACUCUAUCAUTT (forward); 5′-AUGAUAGAGUACAUGGGACTT-3′ (reverse).
Lipofectamine rnaimax lipid reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
Lipofectamine RNAiMAX is a lipid reagent designed for the transfection of small interfering RNA (siRNA) and microRNA (miRNA) in a wide range of cell types, including hard-to-transfect cells. The reagent facilitates the delivery of these RNA molecules into cells to enable efficient gene silencing or regulation.
Automatically generated - may contain errors
Lab products found in correlation
A10001, by GenePharma (1 mentions)
Opti mem medium, by Thermo Fisher Scientific (1 mentions)
Control sirna, by Santa Cruz Biotechnology (1 mentions)
Control sirna, by Thermo Fisher Scientific (1 mentions)
Bcl 2, by Thermo Fisher Scientific (1 mentions)
Sirna, by Thermo Fisher Scientific (1 mentions)
Human ifnα, by Thermo Fisher Scientific (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
10 protocols using lipofectamine rnaimax lipid reagent
1
Transient Transfection of C3 siRNA
2
M-CSF Knockdown in Mouse LECs
3
Modulating nAChR α7 subunit in IRE1α-overexpressing INS-1 cells
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A negative control (Qiagen, #1022563) or two small interfering RNAs (siRNAs) against rat CHRNA7 (Qiagen, #SI00253365, #SI03045028), encoding nAChR α7 subunit, were transfected using lipofectamine RNAiMAX lipid reagent (Invitrogen) in Dox‐inducible IRE1α‐overexpressing INS‐1 cells, in accordance with the manufacturer’s protocol. A total of 24 h after the transfection, cells were treated with Dox with or without nicotine or PNU for 24 h. Then, relative TXNIP or Ins1 mRNA expression levels were determined by quantitative PCR as described above.
4
Modulating Bcl2 family in β and α cells
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β and α cells were transfected with 30 nM of the previously validated siRNAs for Bcl2l1 (Invitrogen, Carlsbad, CA) (Miani et al., 2013 (link)), Bcl2 (Invitrogen, Carlsbad, CA) (Cunha et al., 2012 (link)) or Allstars Negative Control siRNA (siCTRL, Qiagen, used as a negative control) using the Lipofectamine RNAiMAX lipid reagent (Invitrogen). siCTRL does not affect β and α cell gene expression, function or viability ((Moore et al., 2012 (link)) and data not shown). Cells were cultured for 48 h and then exposed to palmitate.
5
STAT1 siRNA Knockdown in α Cells
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α Cells were transfected with 30 nM of the previously validated siRNA for STAT1 (5′-CCCUAGAAGACUUACAAGAUGAAUA-3, Invitrogen, Carlsbad, CA, USA; [Moore et al., 2011 (link)]) or Allstars Negative Control siRNA (siCTRL, Qiagen, Venlo, the Netherlands; used as a negative control) using the Lipofectamine RNAiMAX lipid reagent (Invitrogen, Carlsbad, CA, USA). siCTRL does not affect α cell gene expression, function, or viability (data not shown). Cells were cultured for 48 hr after transfection and then infected with CVB5.
6
Knockdown of AKT1 and GSK3β in Cells
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Cells were transfected with the corresponding small interfering RNA (siRNA) using Lipofectamine™ RNAiMAX lipid reagent (Invitrogen, CA, USA) as per manufacturer’s instructions. Briefly, 2 × 105 cells were plated unicellular on Vitronectin-coated 24-well dishes, grown 24 hours with E8 media and then transfected with Silencer Select Negative Control #2 (Ambion™, cat#4390846), Silencer Select Validated AKT1 siRNA (Ambion™, Cat. # 4390824, siRNA ID:s659) or Silencer Select Validated GSK3β siRNA (Ambion™, Cat. # 4390824, siRNA ID: s6241) (Invitrogen, CA, USA). The concentration of siRNA used for cell transfection was 10 nM.
7
Optimized siRNA and miRNA Inhibitor Transfection
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The short interfering RNAs (siRNAs) and single-stranded miRNA inhibitors used in this study are described in Supplementary Table 3 . The siRNAs targeting DP5 and PUMA have been previously validated, including comparison against a second siRNA causing similar biological effects (34 (link)). The optimal concentration of siRNAs used for cell transfections (30 nmol/L) was previously established by our group (30 (link),35 (link)). Single-stranded miRCURY LNA inhibitors (Exiqon, Vedbaek, Denmark) that specifically block endogenous miRNAs were used at a concentration of 120 nmol/L based our own dose-response experiments (data not shown) and as described (25 (link)). Allstar Negative Control siRNA (siCTRL; Qiagen) was used as negative control in all experiments. This siCTRL does not affect β-cell gene expression or insulin release as compared with nontransfected cells (35 (link)). Transient transfection was performed with Lipofectamine RNAiMAX lipid reagent (Invitrogen-Life Technologies) following the manufacturer’s instruction. After an 8-h transfection, cells were cultured for a 48-h recovery period before exposure to cytokines.
8
siRNA Silencing of YIPF5, CHOP, and DP5
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YIPF5 was silenced using 2 siRNAs targeting different sequences of YIPF5 (si1 SI04182745 and si2 SI04344984, Qiagen). CHOP was silenced using SI3041633 (Qiagen) and DP5 using s194952 (Ambion). Allstar Negative Control siRNA (siCT, Qiagen) was used as negative control. This siRNA does not affect β cell gene expression, function, or viability (54 (link)). Transient transfection was performed using 30 nM siRNA and Lipofectamine RNAiMAX lipid reagent (Invitrogen/Life Technologies) as previously described (54 (link)). After overnight transfection, cells were cultured at least 8 hours before treatment.
9
Gene Silencing and IFNα Exposure in β Cells
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NLRC5 gene expression was silenced using two independent siRNAs [Thermo Fisher Scientific; siNL#1 (siRNA HSS130675): 5′-GGACACCUGGCAGUCUUUCAUUCAU-3′; siNL#2 (siRNA HSS130676): 5′-GCAGUUGGCAGAGUCUCUCGUUCUU-3′]. AllStars Negative Control siRNA (siCTL) (Qiagen) was used as a negative control; the siRNA control does not interfere with β cell gene expression, function, or viability (82 (link)). NOVA1 gene expression was silenced using an siRNA [Thermo Fisher Scientific; siNO1 (siRNA HSS143142): 5′-UUUGCAACUGAACAAUUGUCUGUCC-3′] (44 (link)). Cells were transfected using the Lipofectamine RNAiMAX lipid reagent (Invitrogen, Life Technologies) in Opti-MEM (Gibco, Thermo Fisher Scientific) reduced serum medium, according to the manufacturer’s instructions. EndoC-βH1 was transfected using 30 nM of each siRNA, overnight. Dispersed human islets and SC-derived β-like cells were transfected with 60 nM of each siRNA, also during an overnight incubation period. After transfection, EndoC-βH1, dispersed human islets, and SC-derived β-like cells were kept in culture for a 48-hour recovery period and subsequently exposed, or not, to human IFNα (2000 U/ml; PeproTech) for 8 or 24 hours. These conditions are based not only on previously published time dose-response experiments (8 (link)) but also on time course and dose response experiments presented in this article (fig. S4).
10
Transfection and Overexpression of DEXI in Pancreatic Beta Cells
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The siRNAs targeting rat and human DEXI used in this study are listed in ESM Table 1. The optimal siRNA concentration (30 nmol/l) and conditions for beta cell transfection were previously established [22, (link)27] (link). Cells were transfected using the Lipofectamine RNAiMAX lipid reagent (Invitrogen, Carlsbad, CA, USA) as described [11] (link). After transfection, cells were cultured for a 48 h recovery period and subsequently exposed to intracellular PIC, treated with proinflammatory cytokines IL-1β plus IFNγ, or infected with coxsackievirus B5 (CVB5).
To overexpress DEXI in INS-1E and EndoC-βH1 cells, we used an overexpression plasmid encoding the human DEXI gene under the control of the cytomegalovirus promoter (pCMV-DEXI) (RC207463, Origene, Rockville, MD, USA). Cells were transfected using the Lipofectamine 2000 lipid reagent (Invitrogen). A plasmid containing only the cytomegalovirus promoter (pCMV-Control) was transfected as a negative control of overexpression.
To overexpress DEXI in INS-1E and EndoC-βH1 cells, we used an overexpression plasmid encoding the human DEXI gene under the control of the cytomegalovirus promoter (pCMV-DEXI) (RC207463, Origene, Rockville, MD, USA). Cells were transfected using the Lipofectamine 2000 lipid reagent (Invitrogen). A plasmid containing only the cytomegalovirus promoter (pCMV-Control) was transfected as a negative control of overexpression.
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