NLRP3 inhibitor tool compound MCC950 (CP-456773 sodium salt, PZ0280) and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich. Lipopolysaccharide (LPS) from Escherichia coli (O55:B5; ALX-581-013-L002) was obtained from Enzo. Nigericin (tlrl-nig-5) and ultrapure flagellins from Bacillus subtilis (Fla-BS; tlrl-pbsfla), Pseudomonas aeruginosa (Fla-PA; tlrl-pafla) and Salmonella typhimurium (Fla-ST; tlrl-epstfla-5) were obtained from Invivogen (purity >95% and endotoxin levels of <0.05 EU/µg). Propidium iodide (PI; P3566) and Lipofectamine 2000 (LF2000; 11668030) were obtained from ThermoFisher. Anti-mouse antibodies for mass cytometry were obtained either pre-conjugated to metal isotope or conjugated in-house using the Maxpar Antibody Labeling Kit (Fluidigm) following manufacturer’s instructions.
Lipofectamine 2000 lf2000
Manufactured by Thermo Fisher Scientific
Sourced in United States
Lipofectamine 2000 (LF2000) is a cationic lipid-based transfection reagent used for the delivery of nucleic acids, such as DNA and RNA, into a variety of cell lines. It facilitates the efficient uptake and expression of these molecules within the target cells.
Automatically generated - may contain errors
Lab products found in correlation
Opti mem, by Thermo Fisher Scientific (4 mentions)
Propidium iodide pi p3566, by Thermo Fisher Scientific (1 mentions)
Mcc950, by Merck Group (1 mentions)
Maxpar antibody labeling kit, by Standard BioTools (1 mentions)
Tnf α, by Promega (1 mentions)
Cercosporamide, by Bio-Techne (1 mentions)
Mcf 7, by American Type Culture Collection (1 mentions)
Hek293t, by American Type Culture Collection (1 mentions)
Ebm 2, by Lonza (1 mentions)
Huvecs, by Lonza (1 mentions)
Silencer select sirna, by Thermo Fisher Scientific (1 mentions)
Sulforhodamine b, by Merck Group (1 mentions)
Freedom evo, by Tecan (1 mentions)
Sic001 1nmol, by Merck Group (1 mentions)
Polybrene, by Merck Group (1 mentions)
Hela cell, by American Type Culture Collection (1 mentions)
Pmd18 t, by Thermo Fisher Scientific (1 mentions)
M mlv reverse transcriptase, by Takara Bio (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
10 protocols using lipofectamine 2000 lf2000
1
NLRP3 Inflammasome Activation Toolkit
2
Cell Culture and Kinase Inhibitor Assay
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HEK293T, MCF7 and HeLa cells were purchased from ATCC (Manassas, VA, USA) and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS), 50 U of penicillin G/ml and 50 μg of streptomycin sulfate at 37 °C in 5% CO2. HUVECs were purchased from Lonza and cultured in endothelial cell basal medium (EBM-2; Lonza, Boston, MA, USA) with EGM-2 SingleQuot growth supplements (Lonza, Madison, WI, USA). Cells that were <6 passages were used in this study. TNFα was purchased from Promega (G5421, Madison, WI, USA). The kinase inhibitors used in this study are: p38 kinase (SB202190; Sigma), CKII (TBCA; Calbiochem, San Diego, CA, USA), MSK (SB747651; Tocris Bioscience, Bristol, UK), JNKs (SP600125; Sigma); ERKs (U0126; Sigma) and MNK (CGP57380; Cayman Chemical, Ann Arbor, MI, USA; or cercosporamide; Tocris Bioscience). The siRNAs were purchased from Thermo Scientific (Waltham, MA, USA) and the siRNA target sequences are listed in Supplementary Table 1 . Antibodies used in this study are listed in the Supplementary Table 3 . The transfection reagents DharmaFECT1 (T-2001) and Lipofectamine 2000 (LF2000) were purchased from Thermo Scientific and Invitrogen (Grand Island, NY, USA), respectively.
3
Transfection of Cells with siRNA
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Transfection with different concentrations of SSOs was performed with PepFect14 (Psyclo Peptide, Inc., Shanghai, China) (Ezzat et al., 2011 (link)) or Lipofectamine® 2000 (LF2000) (Life Technologies, Invitrogen, CA, USA) or OptiMEM (gymnotic delivery), which was formulated in OptiMEM for 15 min at room temperature according to the protocols of the manufacturer, and was then added to the cells cultured in DMEM + 10% FBS. The complexes were left in the culture for 24 h, after which the cells were harvested for RNA isolation or luciferase measurement. Transfection with 5 nM of Silencer Select siRNA® (Life Technologies, Carlsbad, CA, USA), including NCL s70420 and TCP1 s224715, was performed with LF2000, formulated in OptiMEM 15 min at room temperature, according to the protocols of the manufacturer, and was then added to the cells cultured in DMEM + 10% FBS. The complexes were left in the culture for 48 h, after which the cells were harvested for RNA isolation.
4
High-Throughput siRNA Screening on Tecan EVO
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Using the Tecan Freedom EVO robotic workstation (Mannedorf, Switzerland), exponentially growing cells were plated in 96-well plates at 30% confluence in 80 μl of medium as recommended by ATCC. Cells were allowed to adhere overnight and then transfected with 20 pmol of a single siRNA library pool suspended in 20 μl OPTI-MEM with 0.1 μl Lipofectamine2000 (LF2000; Life Technologies, Carlsbad, CA, USA). Each 96-well plate contained 80 siRNA pools; 3 negative, non-targeting scrambled control (siSCR) wells; and 3 positive control (siTOX) wells. After overnight incubation, the medium in all wells was changed. For the sensitizer screen, trastuzumab (10 μg/ml) was added to the appropriate wells. At 4 days post transfection, cells were fixed with 10% trichloroacetic acid and stained with sulforhodamine B (Sigma-Aldrich, St Louis, MO, USA) to quantitate total cellular protein content, which was taken as a measure of cell proliferation.44
5
Specific Knockdown of AMOT-130 Isoform
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Isoform-specific siRNA oligonucleotides that target the F-actin–binding domain in rat AMOT-130 were obtained from Ambion/Thermo Fisher Scientific and Sigma-Aldrich. The target sequences were: si130#1, 5′-GTCCGATCCTTGAGCGAAA-3′ (s152896; Ambion), and si130#2, 5′-CCAAGATGAAGGCCTTAGA-3′ (SASI_Rn02_00284990; Sigma-Aldrich). A CsiRNA whose sequence was not homologous to any vertebrate sequence was used as negative control (SIC001-1NMOL; Sigma-Aldrich). To evaluate the knockdown efficacy of endogenous AMOT-130, 20 µM siRNA were transfected to a final concentration of 10 nM in rat IEC-18 cells (passage 5–10) using Lipofectamine 2000 (LF2000; Invitrogen). Cells were harvested 4 d later, and the relative expression levels of endogenous AMOT-130 were analyzed by immunoblotting using anti–AMOT-130 antibody. Quantification of the Western blot membranes by densitometry estimated a reduction in the AMOT-130 levels to ∼90% in IEC-18 cells relative to controls (see Fig. 3 A ). For knockdown experiments, neurons at 11 DIV were transfected with siRNA together with 5 µg pEGFP–β-actin plasmid using LF2000 and prepared for experiments at 15–16 DIV. Alternatively, 2.5 × 106 dissociated hippocampal neurons were electroporated with 1 µM si130#1 or CsiRNA at the day of plating (0 DIV) and analyzed by immunoblotting at 9 DIV.
6
Lentiviral Knockdown of PTEN, DNMT3B, and G9a
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The expression plasmids and the bacteria clone for PTEN shRNA (TRCN0000029738), DNMT3B shRNA (TRCN0000035687 and TRCN0000035688), and G9a (TRCN00000115667 and TRCN00000115668) were provided by the National Science Council in Taiwan. Lentiviral production was performed by transfection of 293T cells using Lipofectamine 2000 (LF2000; Invitrogen, Carlsbad, CA). Forty-eight hours after transfection, supernatants were collected and filtered. Subconfluent cells were infected with lentivirus in the presence of 8 μg/ml polybrene (Sigma-Aldrich). At 24 h post-infection, the medium was replaced with fresh growth medium containing puromycin (1 μg/ml) and selected for infected cells for 48 h.
7
Transient Transfection of HeLa Cells
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HeLa cells (ATCC; American Tissue Culture Collection) were cultured in 100 mm dishes in full medium (DMEM containing 10% fetal bovine serum, non-essential amino acids, high glucose) at 37 °C in 5% CO2. Confluent cells were harvested with 0.05% trypsin/0.02 mM EDTA in PBS, and subcultured at a ratio of 1:2–1:3. Cells were plated onto six-well plates and transfected the following day (70–80% confluence) using Lipofectamine 2000 (LF2000) (Invitrogen, Carlsbad, CA, USA) for 4 h in DMEM, followed by replacement with full medium. Double-transfections were performed using 2 μg of LRRK2 plasmids and 500 ng of RAB plasmids of interest. Alternatively, and where indicated, cells were transfected using Jetprime in full medium overnight according to manufacturer’s instructions using the same DNA concentrations as for transfections with LF2000. The following day, transfected cells were replated at a 1:2 ratio onto coverslips in 24-well plates, and proteins were expressed for 48 h before analysis.
8
Cloning and Transfection of PTEN in Mammary Cells
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Total RNA was extracted from mammary gland tissue and cDNA was generated using M-MLV reverse transcriptase (TaKaRa). Pten-specific primer sequences (sense 5′-GGA ATT CCC GTT CCG AGG ATT ATT C-3′; antisense 5′-GGG GTA CCG TAA AAC AAG ATT GGT CAG G-3′) were used to amplify the desired sequence. After digestion of the PCR products with EcoRI and KpnI, the Pten gene segment was cloned into pMD18-T (Ambion) to generate pMD18-T-Pten. All clones were verified by DNA sequencing. Cloning of the Pten gene segment into pGCMV-IRES-EGFP (Ambion) was conducted using a similar method to that for pMD18-T-Pten.
DCMECs were transfected with the pGCMV-Pten-IRES-EGFP (recombinant plasmid) or pGCMV-IRES-EGFP (empty vector) using Lipofectamine 2000 (LF2000) according to the manufacturer’s recommendations (Invitrogen). Briefly, DCMECs (1×106 cells per well) were plated in 6-well culture plates. For each well, 1 µg of the appropriate plasmid DNA and 2.5 µL of LF2000 was diluted in 200 µL of OPTI-MEMI medium and incubated at room temperature for 20 min to allow for the formation of lipocomplexes; complexes were then added to wells. Cells were incubated with serum- and antibiotic-free medium at 37°C for 36 h. Optimal transfection conditions were screened in advance (Figure S3 ).
DCMECs were transfected with the pGCMV-Pten-IRES-EGFP (recombinant plasmid) or pGCMV-IRES-EGFP (empty vector) using Lipofectamine 2000 (LF2000) according to the manufacturer’s recommendations (Invitrogen). Briefly, DCMECs (1×106 cells per well) were plated in 6-well culture plates. For each well, 1 µg of the appropriate plasmid DNA and 2.5 µL of LF2000 was diluted in 200 µL of OPTI-MEMI medium and incubated at room temperature for 20 min to allow for the formation of lipocomplexes; complexes were then added to wells. Cells were incubated with serum- and antibiotic-free medium at 37°C for 36 h. Optimal transfection conditions were screened in advance (
9
Hornerin Knockdown in Keratinocytes
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For in vitro siRNA-mediated hornerin knockdown, COCA cells and human keratinocytes were plated at a density of 0.1 × 106 cells/well in a six-well cluster plate. The cells were treated the following day with Scr siRNA or Hrnr siRNA (Invitrogen; siRNA sequences identical to those utilized in tumor studies) following an established protocol described here68 (link). Briefly, 100 µL of 10 µM siRNA and 12.5 µL of Lipofectamine 2000 (LF2000) (Invitrogen) were added to separate 625 µL OptiMEM (Thermo Fisher Scientific) aliquots. After 5 min at RT, the solutions were mixed and incubated for an additional 30 min, upon which 5 ml of keratinocyte media were added. The cells were incubated overnight in 3 ml of the siRNA/LF2000/media mixture, replenished the following morning with media containing 2 mM CaCl2 to induce differentiation, and lysed in RIPA buffer 8 h post-media change. Lysate preparations were subjected to immunoblot using the primary antibodies anti-human hornerin (Abcam; (1:1000)), anti-mouse hornerin (Santa Cruz Biotechnology, Dallas, TX, USA, sc164605; (1:200)), or anti-beta actin (Cell Signaling; (1:4000)), and respective secondary antibodies anti-goat HRP (R&D Systems; (1:10,000)), and anti-mouse HRP (R&D Systems; (1:10,000)). Uncropped hornerin immunoblots are represented in Supplementary Fig. 4 .
10
HeLa Cell Transfection Protocol
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HeLa cells were cultured in 100-mm dishes in full medium (DMEM containing 10% fetal bovine serum, non-essential amino acids, high glucose) at 37 ºC in 5% CO 2 .
Confluent cells were harvested with 0.05% trypsin/0.02 mM EDTA in PBS, and were subcultured at a ratio of 1:2 -1:3. Cells were plated onto 6-well plates and transfected the following day (70-80% confluence) using Lipofectamine 2000 (LF2000) (Invitrogen) for 4 h in DMEM, followed by replacement with full medium. Double-transfections were performed using 2 μg of LRRK2 plasmids and 500 ng of RAB plasmids of interest.
Alternatively and where indicated, cells were transfected using Jetprime in full medium overnight according to manufacturer´s instructions using the same DNA concentrations as for transfections with LF2000. The following day, transfected cells were replated at a 1:2 ratio onto coverslips in 24-well plates, and proteins expressed for 48 h before analysis.
Confluent cells were harvested with 0.05% trypsin/0.02 mM EDTA in PBS, and were subcultured at a ratio of 1:2 -1:3. Cells were plated onto 6-well plates and transfected the following day (70-80% confluence) using Lipofectamine 2000 (LF2000) (Invitrogen) for 4 h in DMEM, followed by replacement with full medium. Double-transfections were performed using 2 μg of LRRK2 plasmids and 500 ng of RAB plasmids of interest.
Alternatively and where indicated, cells were transfected using Jetprime in full medium overnight according to manufacturer´s instructions using the same DNA concentrations as for transfections with LF2000. The following day, transfected cells were replated at a 1:2 ratio onto coverslips in 24-well plates, and proteins expressed for 48 h before analysis.
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