Knockdown of A20 in PMs was performed using anti-A20 small interfering RNA (si A20; 5’-GGGUAGGUUUGAAGACUUAtt-3’). We acquired siA20 and the nontargeting control siRNA (scrambled siRNA; siNC) from TsingKe Biological Technology (Beijing, China). Several groups were included: untreated, lipofectamine6000-treated (lipo6000; Beyotime, Shanghai, China), Giardia-treated; lipo6000 + Giardia-treated, siNC-treated, siNC + Giardia-treated, siA20-treated, and siA20 + Giardia-treated. PMs were transfected at 70% confluence using siRNA at a concentration of 50 nM and lipo6000. In brief, siRNA and lipo6000 were diluted separately in OPTI-MEM medium (Gibco, Carlsbad, CA, USA), and then mixed at a 1:1 ratio and incubated for 5 min. At 48 h after siRNA transfection, qPCR and immunoblotting assays were performed to detect the silencing efficiency. Successfully transfected cells were treated with trophozoites for 12 h for further analysis.
Lipo6000
Manufactured by Beyotime
Sourced in China
The Lipo6000 is a laboratory equipment used for the analysis and characterization of lithium-ion batteries. It provides a range of testing and measurement functions to evaluate the performance and safety of lithium-ion battery cells and modules.
Automatically generated - may contain errors
Lab products found in correlation
Opti mem medium, by Thermo Fisher Scientific (2 mentions)
Dual luciferase reporter assay kit, by Promega (2 mentions)
Hygromycin b, by neoFroxx (2 mentions)
Dual luciferase reporter assay system, by Promega (2 mentions)
Si nc, by Tsingke (1 mentions)
Scrambled sirna, by Tsingke (1 mentions)
Si nc, by Beyotime (1 mentions)
Pex 3, by GenePharma (1 mentions)
Dual luciferase assay system, by Promega (1 mentions)
Pmir rb report vector, by RiboBio (1 mentions)
Mir 20b mimic, by RiboBio (1 mentions)
Renilla reniformis luciferase, by Promega (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Hyclone, by GE Healthcare (1 mentions)
Axyprep dna miniprep kit, by Corning (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Control sirna, by RiboBio (1 mentions)
Dual luciferase reporter assay kit, by Beyotime (1 mentions)
43 protocols using lipo6000
1
Silencing A20 in Macrophages Infected with Giardia
2
Modulation of SENP3 Expression in LPS-Induced Renal Inflammation
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The SENP3-siRNA, negative Con (NC)-siRNA, SENP3-plasmid, and NC-plasmid (empty vehicle pEX-3) were purchased from GenePharma (Shanghai, China) and were transfected into NRK-52E cells using the Lipo6000TM (Beyotime) according to the manufacturer's instructions. Then, NRK-52E cells were subjected to LPS addition for the indicated times. Next, cells were lysed for protein extraction, and samples were analyzed by Western blotting. The sequences of SENP3-siRNA oligonucleotides were as follows: sense 5′-GGUACUACAGCUGAUCCAATT-3′ and antisense 5′-UUGGAUCAGCUGUAGUACCTT-3′. The full sequence of SENP3-Plasmid was described in the online supplementary information (see www.karger.com/doi/10.1159/000525308 for all online suppl. material).
3
STAT3 3'UTR Luciferase Assay
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Oligonucleotides needed for the production of the luciferase reporter vector were designed based on the expected binding site of the 3′-untranslated region of the STAT3 mRNA. A DNA fragment that was located in the middle of the expected binding site was subjected to PCR amplification, and the resultant product was inserted into the pmiR-RB-ReportTM vector between the Not-I and Xho-I restriction sites (RiboBio Co., Ltd., Guangzhou, China). Oligonucleotides of the wild-type version (wt) and mutated version (mut) of the predicted binding site were constructed (RiboBio Co., Ltd., Guangzhou, China). 293 T cells were cultured in DMEM and cotransfected with 50 ng of empty reporter (vehicle), pmiR-RB-STAT3-wt or pmiR-RB-STAT3-mut, and 50 nM negative control miRNA, miR-106a mimic (no. miR10000385; RiboBio Co., Ltd., Guangzhou, China), or miR-20b mimic (no. miR10003187; RiboBio Co., Ltd., Guangzhou, China) using Lipo6000TM (Beyotime, China). After a waiting period of twenty-four hours, the cells were tested with the Dual-Luciferase Assay System to determine whether or not they contained firefly luciferase activity and Renilla reniformis luciferase activity (Promega, USA).
4
Nrp1 3'-UTR Luciferase Reporter Assay
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Luciferase reporter assays were performed following the manufacturer's protocol. 293 T cells were seeded in a 24-well plate and co-transfected with luciferase reporter constructs encoding the wild-type 3'-UTR region of Nrp1 (Nrp1-3'-UTR-WT) or a mutated Nrp1 3'-UTR region (Nrp1-3'-UTR-MUT), and miR-26a-5p, miR-129-1-3p, miR-186-5p, miR23b-3p, and miR-365-3p mimics (50nM), or Non-target Control (NC) (Ribobio, Guangzhou, China) using Lipo6000TM (Beyotime). Luciferase activities were measured using a Dual-Luciferase Reporter Assay Kit (Promega, Madison, WI, USA) according to the manufacturer's protocol. Each experiment was performed in triplicate.
5
Transfecting hEM15A Cells with siRNA
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SiRNA provided by Integrated Biotech Solutions company (Shanghai, China). The hEM15A cells were seeded in six-well plates and grown in a 37℃ incubator overnight for transfection when the cell density reached 40%. Transfection reagents Lipo6000TM (Beyotime) was used for cell transfection.
6
Regulation of Cytokine Production by miRNAs
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To determine the role of each miRNA in regulating cytokine production, PMφ was first transfected with individual miRNA inhibitors (10 nM, miR-21a-5p, miR-23b-5p, miR-342-5p, miR-378a-3p, miR-532-5p, miR-6238 or let-7b-5p) using Lipo6000 (C0526, Beyotime, Shanghai, China) for 4 h as previously reported [21 (link)]. After that, these cells were challenged with LPS/γ-IFN with or without M2-EV (10 μg/mL) treatment. At the indicated times after incubation, the changes in cytokines (TNF-α and IL-6) and proinflammatory pathways (NF-κB, JAK-STAT and p38 MAPK) in cells were analyzed.
7
DLEU1 silencing in RCC cell lines
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The human RCC cell lines KETR3 and 786-O were obtained from the Cell Bank of Chinese Academy of Sciences and cultured in DMEM (HyClone; GE Healthcare Life Sciences) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and antibiotics (penicillin, 100 U/ml; streptomycin, 0.1 mg/ml; Sigma-Aldrich; Merck KGaA) at 37°C with 5% CO2. In total, ~6×104 cells were cultured overnight until 60–70% convergence and cells were subsequently transfected with 50 nM small interfering RNA (siRNA/si)-DLEU1 (Oligobio) or scrambled siRNA [as negative control (NC); Oligobio] using Lipo6000 (Beyotime Institute of Biotechnology) and cultured for 6–8 h at 37°C. Subsequently, the medium containing the transfection reagent was removed and the cells were cultured in DMEM for 24–48 h before the next experiments. The sequence of siRNA-DLEU1 was 5′-CAACGGAAUGUAUCAAUGATT-3′, the sequence of siRNA-control was 5′-TTCTCCGAACGTGTCACGT-3′.
8
Investigating let-7a-5p regulation of BCL2L1
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The mature sequences of let-7a-5p mimics, inhibitors, and their corresponding negative controls (listed in below) were designed and synthesized by Sangon Biotech (Shanghai, China). The inserted fragment was validated using DNA sequencing technology. Cell transfection and co-transfection were conducted using Lipo6000 (Beyotime, Shanghai, China). Firefly luciferase reporter plasmids containing 3′ untranslated region (UTR) of the BCL2L1 were purchased from AmyJet Scientific Inc. (Wuhan, China). The mutations were induced with the supposed target site of BCL2L1 3′-UTR using the QuickChange XL kit (Stratagene, La Jolla, CA, USA) according to the manufactures instructions. The primers were:
let-7a-5p mimic: 5′-UGAGGUAGUAGGUUGUAUAGUU-3′
Negative control: 5′-UUCUCCGAACGUGUCACGUTT-3′
let-7a-5p inhibitor: 5′-AACUAUACAACCUACUACCUCA-3′
Negative control : 5′-AGCUCCCAAGAGCCUAACCCGU-3′
For aberrant expression of BCL2L1, the A549 lung cancer cells were transfected with pcDNA3.1 plasmids and shRNAs, respectively. To overexpress BCL2L1, the full length of BCL2L1 cDNA was amplified and subcloned into pcDNA3.1; whereas 3 shRNAs were synthesized to knockdown the expression of BCL2L1. The empty pcDNA3.1 plasmid and a scrambled shRNA were used as negative controls, respectively. All plasmids were isolated and purified using the AxyPrep DNA Miniprep Kit (Axygen Scientific, CA, USA).
let-7a-5p mimic: 5′-UGAGGUAGUAGGUUGUAUAGUU-3′
Negative control: 5′-UUCUCCGAACGUGUCACGUTT-3′
let-7a-5p inhibitor: 5′-AACUAUACAACCUACUACCUCA-3′
Negative control : 5′-AGCUCCCAAGAGCCUAACCCGU-3′
For aberrant expression of BCL2L1, the A549 lung cancer cells were transfected with pcDNA3.1 plasmids and shRNAs, respectively. To overexpress BCL2L1, the full length of BCL2L1 cDNA was amplified and subcloned into pcDNA3.1; whereas 3 shRNAs were synthesized to knockdown the expression of BCL2L1. The empty pcDNA3.1 plasmid and a scrambled shRNA were used as negative controls, respectively. All plasmids were isolated and purified using the AxyPrep DNA Miniprep Kit (Axygen Scientific, CA, USA).
9
Cytokine Regulation by miRNA Inhibitors
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To determine the role of each miRNA in regulating cytokine production, PMφ was firstly transfected with individual miRNA inhibitors (10 nM, miR-21a-5p, miR-23b-5p, miR-342-5p, miR-378a-3p, miR-532-5p, miR-6238 or let-7b-5p) using Lipo6000 (C0526, Beyotime, Shanghai, China) for 4 h as previously reported [23] . After that, these cells were challenged with LPS/γ-IFN with or without M2-EVs (10 μg/ml) treatment. At indicated time after incubation, the changes in cytokines (TNF-α and IL-6) and proinflammatory pathways (NF-κB, JAK-STAT and p38 MAPK) of cells were analyzed.
10
HMGB1 3'UTR Luciferase Assay
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HMGB1 3ʹ untranslated region (UTR) wild-type and mutant reporter plasmids were
designed and synthesized by Guangzhou Ruibo Biotechnology Co., Ltd. (Guangzhou,
China). The cells were grown in 24-well plates, and each group of 3 wells was
further cultured for 24 hours. Transfection was carried out when BEAS-2B
confluency was between 50% and 70%. Cells without plasmids were transfected with
miR-129-5p mimic or miR-NC (negative control) using Lipo6000™ (Beyotime,
Shanghai, China). After culture for an additional 48 hours, luciferase activity
was detected.
designed and synthesized by Guangzhou Ruibo Biotechnology Co., Ltd. (Guangzhou,
China). The cells were grown in 24-well plates, and each group of 3 wells was
further cultured for 24 hours. Transfection was carried out when BEAS-2B
confluency was between 50% and 70%. Cells without plasmids were transfected with
miR-129-5p mimic or miR-NC (negative control) using Lipo6000™ (Beyotime,
Shanghai, China). After culture for an additional 48 hours, luciferase activity
was detected.
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