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.
Si nc
Manufactured by Beyotime
Sourced in China
The Si-NC is a versatile laboratory equipment used for the synthesis and characterization of silicon nanocrystals (Si-NCs). It is designed to provide a controlled environment for the fabrication of these nanomaterials, which have various applications in fields such as optoelectronics, photovoltaics, and biomedical imaging.
Automatically generated - may contain errors
Lab products found in correlation
Si nc, by GenePharma (2 mentions)
Lipo8000 transfection reagent, by Beyotime (2 mentions)
Opti mem medium, by Thermo Fisher Scientific (1 mentions)
Si nc, by Tsingke (1 mentions)
Lipo6000, by Beyotime (1 mentions)
Scrambled sirna, by Tsingke (1 mentions)
Si ampk, by GenePharma (1 mentions)
Lipo6000 transfection reagent, by Beyotime (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Cell counting kit 8 cck 8 assay, by Beyotime (1 mentions)
24 well plate, by Corning (1 mentions)
Opera phenix high content screening system, by PerkinElmer (1 mentions)
Dulbecco s modified eagle s medium, by Beyotime (1 mentions)
5 protocols using si nc
1
Silencing A20 in Macrophages Infected with Giardia
2
AMPK Knockdown in Cell Lines
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The small interfering RNA of AMPK (si-AMPK) and negative control (si-NC) were designed and synthesized by GenePharma Co., Ltd (China). Approximately 1 × 105 cells were seeded per well in a 6-well plate and cultured until reaching 50–60% confluence. Transfection was performed using Lipo8000™ Transfection Reagent (Beyotime, China) along with si-AMPK or si-NC (100 pmol/well). After 12 h, the medium was replaced, and cells were further cultured for an additional 36 h. Then, the cells underwent intervention with ICA (25 µM) for 24 h.
3
RNA Interference in Sea Cucumbers
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circ432 siRNA (si-circ432), AjELMO1 siRNA (si-AjELMO1), miR-2008 mimics/inhibitor (miR-2008M/miR-2008I) and their negative controls (si-NC, NC mimics (NCM), NC inhibitor (NCI) were synthesized by GenePharma Company (Shanghai, China). The detailed sequence information is shown in Table 1 . For in vitro transfection experiments, 1 μL si-circ432, si-AjELMO1, or miR-2008M/miR-2008I and their negative controls (si-NC, NCM, NCI) were mixed with 1 μL Lipo6000 transfection reagent (Beyotime, Shanghai, China). The mixture was then transfected into 500 μL of primary cultured cells in each well and incubated in the dark for 48 h before use at 16 °C. For the in vivo experiment, 10 μL circ432 siRNA, AjELMO1 siRNA or miR-2008M/miR-2008I, and negative control were mixed with 10 μL Lipo6000 transfection reagent and 80 μL PBS as the transfection solution. Sea cucumbers (100 ± 10 g) were infected with 100 μL of the above transfection solution. Control and treated coelomocytes were collected for further RNA and protein extraction 48 h after being treated.
4
Overexpression and Knockdown of PVT1
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The sequences of PVT1 were synthesized and cloned into pcDNA3.1 plasmid to construct the overexpression vector of PVT1 (pcDNA3.1-PVT1) by Beyotime Company (Shanghai, China). Sequences of small interfering RNA of PVT (si-PVT1), si-NC, miR-497-5p mimic or inhibitor, mimic-NC, or inhibitor NC were synthesized and provided by Beyotime Company (Shanghai, China). Cells were seeded in six-well plates and allowed to adhere for 24 h. Then the cell transfection was performed using Lipofectamine 2000 (Invitrogen, Waltham, MA, USA), and the experimental operation was carried out according to the instructions.
5
Cell Proliferation Assay for A549 and H358 Lung Cancer Cells
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A549 and H538 were purchased from the Chinese Academy of Science Cell Bank and cultured in Dulbecco's Modified Eagle's Medium (DMEM Beyotime, CN) supplemented with 10% Fetal bovine serum (FBS, EveryGreen, Zhejiang, China) and 1% antibiotics at 37 °C in 5% CO2. Small interfering RNA (siRNA) and the corresponding negative control (siNC) were purchased from Ribobio (Shanghai, China). A549 and H358 cells were transfected with siDARS2, siCOX5B, and siNC using the Lipo8000 Transfection Reagent (Beyotime Biotechnology, China). A549 cells with GFP fluorescence were obtained as reported previously [29 (link)]. Cell viability assay was also performed using Cell Counting Kit -8 (CCK-8) assay (Beyotime, CN) according to the previous study. The relative cell viability was calculated as follows: Relative Cell Viability = (Test absorbance/Mean absorbance of control wells). At the logarithmic growth phase, 1500 cells were seeded into 24-well plates (Corning, NY, USA) at 100 μL of cell suspension per well. Cell proliferation was measured according to corresponding fluorescence intensity after incubation for 0, 24, 48, 72, 96, and 120 h at 37 °C. Images were acquired on the Opera Phenix High Content Screening System (PerkinElmer) and analyzed on Harmony High-Content Imaging and Analysis Software.
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