p53 siRNA and non-targeting siRNA were purchased from Guangzhou RiboBio Co, Ltd. (Guangzhou, China). For siRNA transfection, cells were seeded in 6-well plates, when they reached 70%-80% confluence, siRNAs were added into the cells with Lipo8000 reagent (Beyotime, Shanghai, China) according to the manufacturer's recommendations. The cells were then exposed to different drugs and harvested for the further experiments including western blot, and biological behavior studies.
Lipo8000 reagent
Manufactured by Beyotime
Sourced in China
The Lipo8000 reagent is a laboratory compound used for the extraction and purification of lipids from various biological samples. It is a versatile reagent designed to efficiently isolate and concentrate lipid components, such as triglycerides, phospholipids, and cholesterol, for further analysis and research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Non targeting sirna, by RiboBio (1 mentions)
P53 sirna, by RiboBio (1 mentions)
Opti mem medium, by Thermo Fisher Scientific (1 mentions)
Dmem medium, by Thermo Fisher Scientific (1 mentions)
Puromycin, by Beyotime (1 mentions)
Gmeasy lentiviral packaging kit, by Genomed (1 mentions)
Hiseq platform, by Illumina (1 mentions)
Truseq pe cluster kit v3 cbot hs, by Illumina (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Geneart site directed mutagenesis system kit, by Thermo Fisher Scientific (1 mentions)
Ha ub, by Addgene (1 mentions)
Dual luciferase reporter gene assay kit, by Yeasen (1 mentions)
Sirt3 sirna, by GenePharma (1 mentions)
Microplate reader, by Agilent Technologies (1 mentions)
Dual luciferase reporter assay kit, by Keygen Biotech (1 mentions)
Tcs sp2, by Leica (1 mentions)
P0126, by Beyotime (1 mentions)
26 protocols using lipo8000 reagent
1
siRNA Knockdown and Drug Testing
2
Modulation of γδ T Cell Function
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Polarized human γδ T cells were cultured in RPMI 1640 medium containing 20% FBS and then transfected with FITC-conjugated control siRNA (Santa Cruz Biotechnology, sc-36869) and IDH2 siRNA (Santa Cruz Biotechnology, sc-62487) with Lipo8000 reagent (Beyotime, C0533). A small amount of FITC-conjugated siRNA was also added into the IDH2 siRNA transfection well. Cells were cultured for additional 48 hours in RPMI 1640 medium with 10% FBS and IL-7 (10 ng/ml). For AGI inhibition assay, single-cell suspensions from oral cancer tissues were treated with varying concentrations of AGI (Sigma-Aldrich) or vehicle control dimethyl sulfoxide for 1 to 2 hours. Cells were stimulated with PMA and ionomycin in the presence of Golgi plug for 4 to 6 hours. Intracellular cytokine staining for IL-17 was performed.
3
Overexpression and Knockdown of Lipid Metabolism Genes
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The STX11, ATGL, LC3B, STX18, and DGAT2 genes were amplified from a mouse (C57BL6) liver cDNA library. The genes were cloned into either pcDNA3.1-Myc/HA, pcDNA3.1-mcherry, or pEGFP-N2 vectors, and transfection was performed using Lipo 8000 reagent (C0533, Beyotime, China). The overexpressing plasmids (pcDNA3.1-Myc/HA, pcDNA3.1-mcherry or pEGFP-N2 vector) and Lipo 8000 reagent were combined carefully in Opti-MEM Medium (11058021, GIBCO, USA) to develop plasmid-lipid complexes. After this, the complexes were added to the cells and incubated for 24 h; the cells were ready for experimentation. The sequences of the STX11 siRNA oligonucleotides (General Biol, China) were as follows: sense 5′-CACUCAAAUUGAAGUAUCATT-3′, and antisense 5′-UGAUACUUCAAUUUGAGUGTT-3′ and were targeted against human STX11. For the STX11 knockdown experiments in HeLa and THLE-2 cells, 100 pmol of mixed oligonucleotides was transfected into cells and plated into 35 mm dishes using Lipo 8000. After three days of incubation, cells were used for the designated assays.
4
Culturing and Transfecting CRC Cells
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Cells used in the study were purchased from American Type Culture Collection and cultured in DMEM medium (Gibco, USA) containing 10% fetal bovine serum (Gibco, USA) and 1% penicillin/streptomycin (Genom, China). Cell lines were reauthenticated with short tandem repeat analysis every 6 months after thawing in our experiments. All cells were incubated in a humidified incubator with 5% CO2 at 37 °C. Expression plasmids for LPCAT2 and LPCAT2 shRNA, PRMT1 shRNA were purchased from GeneCopoeia (Guangzhou, China). Lipo8000 reagent (Beyotime Biotechnology, China) was used for transient transfection. Puromycin (2.5 μg/ml) was utilized to select LPCAT2 overexpression or knockdown CRC cell lines.
5
Cell Culture and Transfection Protocol
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HT-22 and SH-SY5Y cells were cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) and 1% penicillin–streptomycin (HyClone, Logan, UT, USA). All the cells were incubated at 37 °C in a humidified incubator containing 5% CO2. For cell transfection, the cells were transfected with shNC and shJWA plasmids for 48 h using lipo 8000 reagent (Beyotime, Shanghai, China). The shJWA plasmid’s construction was conducted with reference to the previous research conducted in our laboratory [16 (link)].
6
Glioma Cell Line Transfection Protocol
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Gliomas cell lines, U251, T98G, U87, and U118, were obtained from ATCC (American type culture collection) and were cultured with DMEM (VivaCell, Shanghai, China) medium plus 10% fetal bovine serum (FBS, VivaCell, Shanghai, China) and 1 × streptomycin and penicillin (NCM, Suzhou, China) in a humidified cell incubator at 37 °C with 5% CO2. SiRNAs (small interring RNAs), targeting WTAP, IGF2BP1, IGF2BP2, IGF2BP3, and FLOT1, and siNC (negative control) were purchased from Sangon Biotech (Shanghai) Inc (Shanghai, China). The sequences of siRNAs were listed in Supplementary Table S1. pENTER-FLOT1 expression plasmid and control plasmid were obtained WZ Bioscience Inc (Shandong, China). SiRNAs and plasmids and were introduced into gliomas cells with Lipo8000™ reagent (Beyotime, Shanghai, China) following the manufacturer's instruction and a published study [19 ]. Briefly, 10 μl siRNA (20 μM) and 10 μl Lipo8000™ reagent were added into 250 μl of serum-free DMEM. After mixing at room temperature for 20 min, the mixture was added to a pre-seeded 6-well plate with cells. After 6 h, the medium was replaced with fresh complete culture medium.
7
Engineered Cell Lines for Research
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BC cell lines, including MDA-MB-231 (SCSP-5043), MCF-7 (SCSP-531), MDA-MB-468 (TCHu136), and BT549 (TCHu 93), non-tumorigenic breast epithelial cell line MCF-10A (SCSP-575), and HEK293T cells (SCSP-502) were obtained from the Chinese Academy of Sciences (Shanghai, China). The conditions for cell culture were described in a previous study [21 (link)]. All siRNAs and their negative control (si-NC) were synthesized by IBSbio (Shanghai, China) and the sequences were listed in Additional file 1 . The plasmids for GFP-fused wild-type (WT), truncated DHX15 and site-mutated DHX15 [P327E, T421A, N422K, Y485E (GFP-DHX15-MUT)] were purchased from IBSbio (Shanghai, China). SiRNAs and plasmids were transfected by Lipo8000 reagent (Beyotime, Shanghai, China). The lentiviral vector pLV-circRNA-Hygro (HarO Life, Shanghai, China) and pCDH-MSCV-MCS-EF1-GFP-puro (IBSbio, Shanghai, China) were applied to construct overexpression plasmid of circRNF10 and DHX15, respectively. After lentiviral packaging using GMeasy Lentiviral Packaging Kit (Genomeditech, Shanghai, China) and cell infection, the cells were selected by 1 μg/mL puromycin (Beyotime, Shanghai, China) or 100 μg/mL hygromycin B (HarO Life, Shanghai, China), according to the antibiotic resistance.
8
Differential Gene Expression Analysis
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HEK293FT cells were transfected with PE2 and FEN1, LIG1, EXO1d or MLH1dn using lipo8000 reagent (Beyotime). Seventy-two hours after transfection, total RNA was harvested from cells using TRIzol reagent (Thermo Fisher), and purified with RNeasy Mini kit (Qiagen) including on-column DNaseI treatment. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq platform and 125 bp/150 bp paired-end reads were generated. The raw data were trimmed using Trimmomatic. Index of the reference genome was built using STAR and paired-end clean reads were aligned to the reference genome using STAR. RSEM was used to count the reads numbers mapped to each gene. Differential expression analysis of two groups was performed using the DESeq2 R package. Genes with an adjusted P-value <0.05 found by DESeq2 were assigned as differentially expressed. The results were plotted using ggplot in R.
9
Transfection and BPA Exposure Assay
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The pIRESpuro3‐EGFP‐Flag‐6 × His‐BRAF‐T1799A plasmid and the control plasmid pIRESpuro3‐EGFP‐Flag‐6 × His (OBio, Shanghai, China) were transfected by Lipo8000™ reagent (Beyotime, China) according to the manufacturer's instructions. After 48 hours transfection, the medium was replaced with different concentrations of BPA‐containing complete medium and the cells were further detected for the following biological function tests.
10
Transient Transfection and EDA-A1 CM Preparation
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HaCaT or HEK293 cells were cultured in DMEM containing 10% FBS and maintained in an incubator containing 5% CO2 at 37 °C. Transient transfection in HaCaT or HEK293 cells with plasmids or SNAP23 siRNA were carried out using Lipo8000™ reagent (Beyotime) by following the manufacturer’s instruction. EDA-A1 CM was prepared as described previously [14 (link)]. Briefly, HEK293 cells (2 × 108) were transfected with 100 µg of EDA-A1 expressing plasmid for 16 h and then changed to culture in DMEM containing 1% FBS. After 24 h, the medium was collected and concentrated tenfold using a Centricon Plus-10 filter (Millipore) and stored at − 80 °C until use.
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