C2C12 myoblasts were obtained from the Stem Cell Bank, Chinese Academy of Sciences, China (Shanghai, China). High purity hydrogen peroxide (H2O2) (30%, 10011218, China) was purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). TMAO of 95% purity was purchased from Sigma-Aldrich (St. Louis, MO, USA) (317594-5G). The GSH-Px assay kit (A005-1-2) was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Nrf2 small interfering RNA (siRNA) was provided by Santa Cruz Biotechnology, Inc. (sc-37049, Dallas, TX, USA). Lipofectamine 2000 (lip2000) for transfection was purchased from Thermo Fisher Scientific (68019, Waltham, MA, USA). Antibodies used in this study include Nrf2 (#12721, CST, USA), HO-1 (#43966, CST, USA), NQO1 (#62262, CST, USA), CAT (21260-1-AP, Proteintech, Wuhan, China), and GAPDH (10494-1-AP, Proteintech, China).
Lipofectamine 2000 lip2000
Manufactured by Thermo Fisher Scientific
Sourced in United States
Lipofectamine 2000 (Lip2000) is a cationic lipid-based transfection reagent used for the delivery of nucleic acids, such as DNA and RNA, into eukaryotic cells. It facilitates the uptake of genetic material into cells by forming complexes with the nucleic acids.
Automatically generated - may contain errors
Lab products found in correlation
Cat 21260 1 ap, by Proteintech (1 mentions)
Nrf2 small interfering rna sirna, by Santa Cruz Biotechnology (1 mentions)
Gapdh 10494 1 ap, by Proteintech (1 mentions)
Gsh px assay kit, by Nanjing Jiancheng (1 mentions)
Hoechst 33342 staining solution, by Beyotime (1 mentions)
Hek293t, by Procell (1 mentions)
Hepg2, by Procell (1 mentions)
Kanamycin, by Solarbio (1 mentions)
Antibiotic antimycotic solution, by Solarbio (1 mentions)
Antifade mounting medium with dapi, by Solarbio (1 mentions)
Cell counting kit 8 cck 8 reagent, by Dojindo Laboratories (1 mentions)
Cucl2, by J&K Scientific (1 mentions)
Bicinchoninic acid bca reagent, by Beyotime (1 mentions)
Seahorse xf glycolysis stress test, by Agilent Technologies (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Deadend fluorometric tunel system, by Promega (1 mentions)
Puromycin, by Solarbio (1 mentions)
Dimethyl sulfoxide (dmso), by Solarbio (1 mentions)
5 protocols using lipofectamine 2000 lip2000
1
C2C12 Myoblast Oxidative Stress Response
2
Comprehensive Cell Culture Protocol
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All cells, including HepG2 (HCC), Huh7 (HCC), HEK-293T (Human embryonic kidney cell), AGS, DLD1, HeLa, and H460, were purchased from Procell Life Science&Technology Co., Ltd (Wuhan, China). HepG2, Huh7, 293T, and HeLa cells were cultured in DMEM (BI, Jerusalem, Israel) medium supplemented with 10% fetal bovine serum (FBS, BI, Jerusalem, Israel). AGS, DLD1, and H460 cells were maintained in RPMI-1640 (BI, Jerusalem, Israel) with 10% fetal bovine serum (BI, Jerusalem, Israel). All cells were grown in a humidified cell incubator at 37 °C with 5% CO2.
Lipofectamine™ 2000 (Lip2000) and Lipofectamine™ 3000 (Lipo3000) were purchased from Thermo Fisher (CA, USA). PEI 40000 was purchased from BIOHUB (Shanghai, China). Actinomycin D and puromycin were obtained from MCE (Shanghai, China). Hoechst 33342 Staining Solution was obtained from Beyotime (Shanghai, China). siRNAs (small interfering RNAs), including siNC and siGAPDH, were synthesized by Sangon Biotech (Shanghai, China). pEGFP-N1 plasmids were purchased from HonorGene (Changsha, Hunan).
Lipofectamine™ 2000 (Lip2000) and Lipofectamine™ 3000 (Lipo3000) were purchased from Thermo Fisher (CA, USA). PEI 40000 was purchased from BIOHUB (Shanghai, China). Actinomycin D and puromycin were obtained from MCE (Shanghai, China). Hoechst 33342 Staining Solution was obtained from Beyotime (Shanghai, China). siRNAs (small interfering RNAs), including siNC and siGAPDH, were synthesized by Sangon Biotech (Shanghai, China). pEGFP-N1 plasmids were purchased from HonorGene (Changsha, Hunan).
3
Cytotoxicity and Apoptosis Assays
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RPMI 1640 medium, fetal bovine serum (FBS), and trypsin-EDTA were purchased from Gibco, USA. Minimum Eagle’s medium (MEM) was obtained from HyClone, USA. Antibiotic/antimycotic solution, kanamycin, puromycin, and dimethyl sulfoxide (DMSO) were obtained from SolarBio, China. DSF was purchased from Selleck, China, whereas CuCl2 was obtained from J&K, China. Phenylmethanesulfonyl fluoride (PMSF), radioimmunoprecipitation assay (RIPA) buffer, and bicinchoninic acid (BCA) reagent were obtained from Beyotime, China. Cell counting kit-8 (CCK-8) reagent was purchased from Dojindo, Japan, whereas AnnexinV-FITC (AV-FITC), propidium iodide (PI) and PI kit were purchased from Roche, Germany. Electrochemiluminescence (ECL) was obtained from Biosharp, China. Lipofectamine 2000 (Lip2000) was purchased from Invitrogen, USA, whereas ROS assay kit was obtained from NJJCBIO, China. Seahorse XF Glycolysis Stress Test and XF Cell Mito Stress Test kits were purchased from Agilent, USA. DeadEnd™ Fluorometric TUNEL System was obtained from Promega, USA, whereas antifade mounting medium with DAPI was purchased from SolarBio, China.
4
Quantitative Protein Analysis of Glioma Cell Lines
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U87 and U251 cells were purchased from the Chinese Academy of Sciences Committee Type Culture Collection Cell Bank (Shanghai, China). Cells were cultured in RPIM 1640 (Life Technologies) supplemented with 10% fetal bovine serum (Millipore). Small interfering RNAs (siRNAs) were purchased from RIBOBIO (Guangzhou, China). Lipofectamine™ 2000 (Lip2000) was obtained from Invitrogen (Carlsbad, CA) and transfections were performed according to the manufacturer’s instructions. The siRNA sequence was as follows: 5ʹ-CCAGAGGUCA GCUGUUCAAGA-3ʹ; Scramble siRNA sequence: 5ʹ-UUCGUAUCUGGGUGUAC CCTT-3ʹ. RIPA lysate was used to extract total protein from the cell lines and the protein concentration in the cell lysate was determined using the BCA protein quantification kit (Beyotime, China). SDS–PAGE was performed in 10% gel with the loading of an equal amount of protein per lane. Samples were electrophoretically transferred onto the PVDF membrane. The membranes were washed five times for 10 min with TBST and blocked using 5% non-fat dry skim milk in TBST before reblotting. The membranes were incubated overnight with primary antibodies, followed by incubation with relevant biotinylated secondary antibodies for 1 hour at room temperature. The membrane was scanned using an infrared Odyssey scanner by Li-cor.
5
Lipofectamine-Mediated Transfection of miRNA and GABRA1
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Lipofectamine 2000 (LIP2000, invitrogen, USA) was applied to transiently transfect the miR-139-5p mimics or inhibitor as well as the GABRA1 overexpression vector or GABRA1 siRNA into glioma cells according to the manufacture’s instruction. MiR-139-5p mimic, miRNA mimic negative control (mimic NC), miR-139-5p inhibitor control, miRNA inhibitor negative control (inhibitor NC) were chemically synthesized by HonorGene (China). GABRA1 overexpression plasmid and siRNA targeting GABRA1 were purchased from RiboBio (China).
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