Small hairpin interfering RNAs (shRNA) against TINCR (shTINCR), TINCR and nontargeting control shRNA (scramble) used a pcDNA3.1-CMV-GFP plasmid as backbone (Clontech, Mountain View, CA, USA). Cells were plated into six-well plates at a density of 5 × 105 cells per well. Cell transfection was conducted with Lipofectamine 2000 (Invitrogen, Carlsbad, USA) according to the protocol recommended by the manufacturer. For in vivo xenograft experiments, plasmids that co-express luciferase and TINCR/shTINCR/scramble were used to prepare lentiviral vectors. Seeded HEK293T cells (ATCC) were co-transfected with 5 μg of prepared plasmids and 5 μg each of packaging plasmids (REV, pMDL and VSV-G) by Lipofectamine-2000. The supernatant was removed 48 hours after transfection and filtered through the 0.45 μm syringe filter, after which the lentivirus in supernatant was further processed, isolated and titrated. For in vitro transduction, a multiplicity of infection (MOI) of 100 was used and the incubation time was 48 hours to allow completeness of viral infection.
Lipofectamine 2000
Manufactured by American Type Culture Collection
Sourced in United States
Lipofectamine 2000 is a cationic lipid-based transfection reagent designed for efficient delivery of DNA, RNA, and other macromolecules into a variety of mammalian cell lines. It is a widely used tool for gene expression studies, gene silencing, and other genetic manipulations in cell culture.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (6 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Hek293 cell, by American Type Culture Collection (2 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions)
Pcdna3.1 cmv gfp plasmid, by Takara Bio (1 mentions)
Polybrene, by Thermo Fisher Scientific (1 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (1 mentions)
Pspax2, by Addgene (1 mentions)
Pmd2 g, by Addgene (1 mentions)
Lipofectamine 2000, by Genechem (1 mentions)
Blasticidin, by Thermo Fisher Scientific (1 mentions)
Phoenix ampho cells, by American Type Culture Collection (1 mentions)
Polybrene, by Merck Group (1 mentions)
Puromycin, by Merck Group (1 mentions)
Genejuice, by Merck Group (1 mentions)
Hela cell, by American Type Culture Collection (1 mentions)
Protein g hrp conjugate, by Bio-Rad (1 mentions)
Luciferase reporter gene assay kit, by Promega (1 mentions)
293t cell, by American Type Culture Collection (1 mentions)
Pgl3 vector, by Promega (1 mentions)
19 protocols using lipofectamine 2000
1
TINCR Knockdown via shRNA Lentiviral Transduction
2
Transduction of ADMSCs with GFP Lentivirus
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Rat ADMSCs were purchased from Procell Life Science & Technology Co., Ltd. and the cells were transplanted into a 25-cm2 plate with DMEM/F12 medium (Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Thermo Fisher Scientific, Inc.), 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in an incubator filled with 5% CO2. LVC-GFP lentivirus vectors were co-transfected with packaging vector psPAX2 (0.1 µg; Addgene, Inc.) and envelope vector pMD2.G (0.9 µg; Addgene, Inc.) (lentiviral plasmid:packaging vector:envelope vector, 10:1:9) into 3rd generation 293T (CRL-3216; ATCC) cells using Lipofectamine® 2000 (cat. no. 11668-019; Invitrogen; Thermo Fisher Scientific, Inc.). Following transfection at 25°C for 48 h, the lentiviral particles were collected via ultracentrifugation at 55,000 × g, at 4°C for 2.5 h. After 24 h of transfection, the cells were cultured for 24 h with serum-free transfer solution as the complete medium. Cells were plated in 6-well plates (1×106 cells/ml) and incubated with LVC-GFP lentivirus (MOI=10) under Polybrene (Thermo Fisher Scientific, Inc.) (5 µg/ml) using Lipofectamine 2000 at 37°C for 24 h. The LVC-GFP lentivirus was synthesized and purchased from Shanghai Genechem Co., Ltd. The medium was then replaced by fresh medium and cultured for another 48 h.
3
Generating Stable GPR37-eGFP N2a Cell Lines
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To generate stable cell lines, we transfected wild-type N2a cells (N2aWT) from ATCC using Lipofectamine 2000 with either eGFP- or GPR37-eGFP-expressing plasmids to generate N2aeGFP and N2aGPR37-eGFP, respectively. Twenty-four hours post-transfection, selection with 500 µg/mL G418 started, and after control cells died, single clones were selected. Both HEK293T and N2a cells were propagated in DMEM with 10% FBS, 1% penicillin–streptomycin, 1x NEAA, 1x GlutaMAX, 1 mM HEPES, and 1x sodium pyruvate. All products for cell culture were purchased from ThermoFisher (Waltham, USA). Imaging confirming expression and trafficking of GPR37-eGFP construct can be found in [43 (link)]. qPCR experiments showed an average of 100-fold increase of GPR37 transcripts in N2aGPR37-eGFP compared to N2aeGFP.
4
Lentiviral and Retroviral Transduction in HEK293T
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Lentiviral plasmids were transfected into HEK293T cells together with packaging and envelope plasmids (psPAX2 and pMD2.G) using Lipofectamine2000 (Thermo Fisher Scientific, Cat#: 11668027) or Genejuice (EMD Millipore, Cat# 70967). At 2 days after transfection, the medium was passed through a 0.45 μm pore filter and mixed with Polybrene (Sigma-Aldrich, Cat#: H9268). The medium containing lentiviruses was transferred to the recipient cells. HEK293T cells were further cultured in fresh medium for 24 hr. After 6 hr of infection, medium was changed. Next day, infection was repeated as above. After lentivirus infection, cells were selected with 1 μg/ml of Puromycin (Sigma-Aldrich, Cat#: P9620) or 5 μg/ml of Blasticidin (Thermo Fisher Scientific, Cat# R21001) for 5-10 days. For retrovirus production, retroviral plasmids were transfected into Phoenix-AMPHO cells (ATCC, Cat#: CRL-3213) using Lipofectamine2000 or Genejuice. Retrovirus infection was carried out as described above.
5
Immunoprecipitation Analysis of SUMOylation
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To analyze SUMOylation within cells, we used Lipofectamine 2000 to transfect HeLa cells (ATCC CRL-2, Manassas, VA) with plasmids encoding wild-type HA-TGM2 along with either SUMO1, SUMO2 and SUMO3 plasmids respectively. Cells were selected and grown according to a previously described protocol [28 (link)]. Cell lysates were immunoprecipitated with a HA-specific affinity matrix gel (Roche, Pleasanton, CA), after which immunoprecipitate was washed in lysis buffer (50 mM Tris-Cl pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 1 mM DTT, 10% glycerol, 10 mM EDTA), followed by elution and proteins resolved by SDS-PAGE and immunoblotted using TG2 antibody, RanGAP antibody (Cell Signal, cat# 36067), and protein G HRP-conjugate (BioRad, Hercules, CA).
6
Validating miR-96 Targeting of Smad7
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TargetScan Human 7.2 (www.targetscan.org/vert_72 ) was used to predict Smad7 as a target of miR-96. The wild-type (wt) or mutant (mut) Smad7 3′-untranslated region (UTR) was cloned into the pGL3 vector (Promega Corporation, Madison, WI, USA). Subsequently, the pGL3-Smad7-3′UTR-wt or pGL3-Smad7-3′UTR-mut vector, along with the miR-96 mimic or the mimic-control (as described above), were transfected with Lipofectamine® 2000 into 293T cells (ATCC, Rockville, MD, USA). Following 48 h, the luciferase activity was measured with a Luciferase Reporter Gene Assay kit (Promega Corporation), using a GloMax-Multi Jr Single Tube Multimode Reader (Promega Corporation). Renilla luciferase activity was used for normalization of the firefly luciferase activity (18 (link)).
7
hGLUT2 Expression in MDCK Cells
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hGLUT2 coding sequence was amplified from HepG2 hepatoma cells (ATCC) and cloned into XhoI and BamHI restriction sites of pmCherry C1 vector containing G418 resistance cassette (Clonetech, Mountain View, CA). MDCK type II cells (ATCC) were transfected using Lipofectamine 2000 according to manufacturer's protocol and maintained under G418 antibiotic selection. Cells were cultured in DMEM culture medium supplemented with 10% FBS, penicillin/streptomycin, non-essential amino acids and l -alanine-l -glutamine. All cells were cultured under standard conditions (i.e. 37°C in a humidified incubator under 5% CO2).
8
Expanding Genetic Code in Mammalian Cells
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Human embryonic kidney (HEK) 293 cells (Sigma-Aldrich) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C with 5% CO2. Cells were co-transfected with 25 μg of plasmids pAckRS-tRNA and pGFPamber-tRNA using Lipofectamine 2000 (Invitrogen) at 80–90% confluence in 100-mm dishes. After 8 h of incubation, medium was replaced with fresh DMEM supplemented with 10% FBS and 10 mM UAA Nɛ-acetyl-lysine (AcK), Nɛ-trifluoroacetyl-lysine (tfAcK) or 3-bromo-phenylalanine (BrF). Cells were collected after 40 h of incubation. NIH3T3 cells (ATCC) were seeded into 100-mm dish and grown in DMEM supplemented with 10% FBS for 24 h. At ∼50% confluence, cells were transfected with 40 μg of plasmids pAckRS-tRNA and pGFPamber-tRNA using Lipofectamine 2000. After overnight incubation, medium was replaced with fresh DMEM containing 10% FBS and 10 mM UAA. Cells were collected 48 h after transfection.
9
High-Density Cortical Neuron Culture Protocols
10
Inflammasome Activation and Bacterial Infection Assay
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Primary BMDMs or THP‐1 cells were seeded in the culture dishes overnight. Cells were primed for 4 h with 200 ng/ml LPS (Sigma‐Aldrich, L9274) and then stimulated as follows: 5 mM ATP (Sigma‐Aldrich, A6419) for 30–45 min, 15 μM nigericin (Invivogen, tlrl‐nig) for 30 min ~1 h respectively. Cells were stimulated with 250 ng/ml FLA‐ST (Invivogen, tlrl‐stfla) for 4 h. Cells were transfected with 2 μg/ml poly(I:C) (Invivogen, tlrl‐pic) or 2 μg/ml poly(dA:dT) (Invivogen, tlrl‐patn‐1) by using Lipofectamine 2000 for 6 h. Citrobacter rodentium (ATCC, 51459), Pseudomonas aeruginosa (ATCC, BAA‐1744), and Salmonella typhimurium strain (ATCC, 14028) grown in lysogeny broth in a log phase were resuspended in phosphate‐buffered saline. For the bacterial infection, each bacteria titer was examined in serial dilution inoculating LB Agar plates. Primary BMDMs were seeded (1 × 106 cells/well) in 12‐well culture plates and allowed to rest for 12 h. Cells were then stimulated with the indicated bacteria.
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