HT22 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Invitrogen, Karlsruhe, Germany), 2 mM L-glutamine and 20 mM HEPES, 100 U/mL penicillin and 100 µg/mL streptomycin (Invitrogen) in 10 cm culture dishes at 37 °C in a humidified incubator containing 5% CO2. HEK293T cells were grown in DMEM/F-12 supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin and 100 µg/mL streptomycin. Passaging of cells took place every 3–4 days with 0.25% trypsin (Invitrogen) for HT22 or without trypsin for HEK293T cells, at a confluency of 75–100%. For in situ PLA and calcium imaging, 1 × 105 HT22 or 2 × 105 HEK293T cells were seeded on Poly-L-lysine coated 20 mm glass coverslips (LaCon, Erbach, Germany) in serum-reduced media (2% FBS). After 24 h at 37 °C, cells were transfected using serum-free media and Lipofectamine LTX Reagent with PLUS Reagent (Invitrogen) at a 3:1 transfection reagent to cDNA ratio. Per coverslip and receptor, 1 µg of cDNA was used.
Lipofectamine ltx reagent with plus reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
Lipofectamine LTX Reagent with PLUS Reagent is a transfection reagent designed to facilitate the introduction of nucleic acids, such as plasmid DNA or siRNA, into a variety of mammalian cell lines. It is a lipid-based reagent that forms complexes with the nucleic acids, enabling their efficient delivery into the cells.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
Dulbecco modified eagle medium (dmem), by Nacalai Tesque (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Glutamax, by Thermo Fisher Scientific (3 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin glutamine solution, by Merck Group (3 mentions)
L glutamine, by Thermo Fisher Scientific (3 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (3 mentions)
Dmem medium, by Thermo Fisher Scientific (3 mentions)
Gps solution, by Merck Group (3 mentions)
Opti mem medium, by Thermo Fisher Scientific (2 mentions)
Bright glo luciferase assay system, by Promega (2 mentions)
Dual glo luciferase assay system, by Promega (2 mentions)
Penicillin streptomycin, by Merck Group (2 mentions)
Leibovitz s l 15 medium, by Thermo Fisher Scientific (2 mentions)
In fusion hd cloning kit, by Takara Bio (2 mentions)
Mccoy s 5a, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
51 protocols using lipofectamine ltx reagent with plus reagent
1
Cell Culture and Transfection Protocol
2
Isolation and Purification of PMCA
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[γ-32P]ATP was obtained from Perkin-ElmerNEN Life Sciences (USA). HEK293T cells were obtained from American Type Culture Collection (ATCC, Manassas, VA). Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum (FBS), Lipofectamine LTX Reagent with PLUS Reagent and cell culture supplements were purchased from Invitrogen-Thermo Fisher Scientific (Carlsbad, CA, USA). Antibiotic/antimycotic solution was obtained from Life Technologies Inc. (Carlsbad, CA, USA). Fluo-4-AM probe was obtained from Molecular Probes (Eugene, OR, USA). PVDF blot membrane was obtained from BioRad Laboratories (Reinach, Switzerland). Dimyristoyl phosphatidylcholine (DMPC) was obtained from Avanti Lipids (Alabaster, AL, USA). Thapsigargin (TG), (-)-epigallocatechin 3-gallate (EGCG), (-)-epicatechin, (+)-catechin, gallic acid, polyoxyethylene (10) lauryl ether (C12E10) and all the other chemicals used in this work were of analytical grade and were purchased from Sigma-Aldrich (St. Louis, MO, USA). Human blood recently drawn at Fundación Fundosol (Buenos Aires, Argentina) was used for the isolation of PMCA. Donors provided informed consent for the donation of blood and for its subsequent legitimate use by the transfusion service.
3
CRISPR-Cas9 Editing in HEK293T and HeLa
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HEK293T (CRL-3216) and HeLa (CCL-2) cells were obtained from ATCC and cultured at 37 °C under humidified 95% air/5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) containing glucose (4.5 g/l), 1 mM sodium pyruvate, 2 mM glutamine and 10% fetal bovine serum (FBS). Cells were seeded at 60% confluence in 24-well plates and transfected after 24 h with 500 ng of DNA in OptiMEM media (Gibco, Life Technologies) using a 1:1 mix of two transfection reagents (Lipofectamine LTX Reagent with Plus Reagent, Invitrogen, and FuGene HD, Promega)30 (link). Each Cas9 variant was transfected in triplicate. For HDR experiments, a short single-stranded homologous DNA was co-transfected in a total amount of 500 ng of DNA including Cas9 plasmid. 72 h after transfection the cells were trypsinized and the cell pellets lysed for DNA extraction using the KAPA Express Extract Kit, according to the manufacturer’s instructions (Sigma-Aldrich). Amplicons were generated using primers flanking the gRNA and incorporating Illumina adaptor sequences (Supplementary Table 2 ). For long amplicon deep sequencing, PCR produced were converted into sequencing libraries by tagmentation (Illumina). Libraries were sequenced on an Illumina MiSeq using 250 bp paired-end chemistry by the Australian Genomics Research Facility (AGRF), Perth, Western Australia.
4
CD22c-EGFP Fusion Protein Expression
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The full-length CD22c coding sequence (aa 1–848) with a Xho I restriction site at the 5′ end and a Bgl II restriction site at the 3′ end was produced by GeneCust laboratories (Luxembourg) and cloned into the pCR™ 2.1-TOPOR plasmid using the TOPO™ TA Cloning™ Kit (Invitrogen, ref K456001). The Xbo I-Xba I CD22c coding sequence was then inserted in pEGFP-N3 (Clontech) and digested by Xho I and Bgl II in order to merge the CD22c and EGFP coding sequences. The CD22c-EGFP coding sequence in pEGFP-N3 and the expression vector pKCR6 were then digested with Xho I and Xba I. The fragments of interest were purified and ligated. The pKCR6/CD22c-EGFP construct was then transfected into Chinese hamster ovary (CHO) cells using the Lipofectamine™ LTX Reagent with PLUS™ Reagent (Invitrogen, ref 15338030).
The transfected cell line was subcloned by limiting dilution and the clones with positive green fluorescence were selected by flow cytometry analysis. Cell lysates from the highest expressing clones were further analyzed with Western blot using an anti-EGFP antibody (B-2). The clones with a positive band at the expected size corresponding to CD22c-EGFP were amplified and used thereafter for hybridoma supernatant screening.
The transfected cell line was subcloned by limiting dilution and the clones with positive green fluorescence were selected by flow cytometry analysis. Cell lysates from the highest expressing clones were further analyzed with Western blot using an anti-EGFP antibody (B-2). The clones with a positive band at the expected size corresponding to CD22c-EGFP were amplified and used thereafter for hybridoma supernatant screening.
5
Analyzing Wnt Signaling in A549 Cells
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The TOP/FOP flash assay was performed based on the protocol [30 (link)]. A549 cells were seeded in 96-well plate. When confluent, cells were transfected with 100 ng/well of either TOP luciferase reporter plasmid or the negative control FOP plasmid using Lipofectamine™ LTX Reagent with PLUS™ Reagent (Invitrogen, Carlsbad, US) in serum-free Opti-MEM® medium (Life Technologies, Carlsbad, US). After 5 h transfection, cells were stimulated with either vehicle, WNT-5A (50 ng/mL), or WNT-5B (50 ng/mL) in Opti-MEM® medium supplemented with 0.1% FBS. After stimulation, cells were lysed by the Bright-Glo™ Luciferase Assay System (Promega). Subsequently, the luciferase activity was measured using a Synergy HTX Multi-Mode Microplate Reader (BioTek, Winooski, US). Data were collected with Gen5 software (BioTek).
6
Purification and Analysis of Sirt7 Protein
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HeLa cells were grown in Dulbecco’s modified Eagle’s medium with 10% FBS, GlutaMAX (1×), 100 U/mL penicillin, and 100 µg/mL streptomycin. Transfection was carried out with Lipofectamine LTX Reagent with Plus Reagent (Invitrogen, cat. #15338100) according to the manufacturer’s instructions. After 48 h, FLAG-tagged Sirt7 (WT) and the corresponding mutants (DM, CA) were collected and lysed in lysis buffer (50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, 10% glycerol and 1 mM DTT) supplemented with protease inhibitors (Roche, cat. #11836153001). The resulting lysate was incubated with anti-FLAG M2 magnetic beads (Sigma, cat. #8823) at 4 °C for 2 h, and the beads were washed with lysis buffer five times, and Sirt7 buffer (50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 5 mM DTT, and 20% glycerol) three times, sequentially. The immobilized proteins were eluted with 3 × FLAG peptide, and ultrafiltration was carried out using using an Amicon Ultra-0.5 10 K device (Merck Millipore, cat. #UFC501024) to concentrate the eluted proteins.
7
Methylation-dependent gene regulation assay
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The gene regions between 500 bp upstream and downstream from the TSSs of Hist1h2ba, Sycp1, and Taf7l were amplified and sequenced. The primer sequences are shown in Supplementary file 5 . Each sequence was cloned into the CpG-free pCpGL-basic Luciferase vector (Klug and Rehli, 2006 (link)). Luciferase reporter constructs were either mock-treated or methylated in vitro with SssI CpG methyltransferase for 4 hr at 37°C and purified with the QIAquick Purification Kit (QIAGEN 28704). Reporter plasmid (500 ng) and Renilla phRL-TK control vector (50 ng; Promega E2241) were co-transfected into HEK293T cells cultured in DMEM containing 10% FBS using Lipofectamine LTX Reagent with PLUS Reagent (Invitrogen 15338100). After 48 hr, cells were lysed, and the relative luciferase activities were analyzed using the Dual-Luciferase Reporter Assay System (Promega E1910) on a Lumat LB 9507 (Berthold). Firefly luciferase (Luc) activity of individual transfections was normalized against Renilla luciferase activity. We analyzed data from three independent experiments to evaluate statistically significant differences.
8
Zeb1 Interactome Identification using BioID
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Zeb1 was cloned from the pLenti-GIII-CMV-hZEB1-GFP-2A-Puro lentiviral vector (Applied Biological Materials Inc., LV362466, Accession No. BC112392) using PCR amplified with primers containing AscI and Not1 (N-terminus tag) or Kpn1 and NotI (C-terminus tag) restriction enzyme sites and cloned in to the pcDNA5 FRT/TO Flag-BirAR118G (pcDNA5 Flag-BirA*). BioID conducted as previously reported52 (link). The N-terminus or C-terminus Flag-BirA tag vector control and Zeb1 were transfected into HEK/293 Flp-In along with pOG44 Flp-Recombinase expression vector using Lipofectamine® LTX Reagent with PLUS™ Reagent as per the manufacturer’s instructions (Invitrogen, Cat. No. 15338100). Cell lines were cultured until colonies were ~3 mm in diameter at before divided into two pools. These were considered as a biological replicate and were processed independently.
Subsequently, all cell lines were cultured in four different conditions and harvested:
Subsequently, all cell lines were cultured in four different conditions and harvested:
DMEM
DMEM + 5 μM MG-132
DMEM + 1 μg/ml Tetracycline and 50 μM biotin
DMEM + 1 μg/ml Tetracycline, 50 μM biotin, and 5 μM MG-132
9
Transfection of MCF7 and HeLa cells
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Transfection was performed using Lipofectamine LTX Reagent with PLUS Reagent (Invitrogen, Carlsbad, CA, USA) or Metafectene Pro from Biontex (München, DE) according to the manufacturer’s instructions. When not indicated cells were processed 24 h after the transfection. HA-V1G1 and HA-RAB7 constructs used in this study have been described previously7 (link),63 (link).
The pCDNA3_2xHA was generated by cloning the fragment of 2xHA 5′ TAC CCA TAC GAT GTT CCG GAT TAC GCT TAC CCA TAC GAT GTT CCG GAT TAC GCT 3′, flanked by a sequence containing the site for KpnI enzyme, into the vector pCDNA3 (Invitrogen).
MCF7 and HeLa cells were silenced with small interfering RNAs (siRNAs) purchased from Eurofins Genomics (Ebersberg, Germany). We used the following oligonucleotides: control RNA, sense 5′-ACUUCGAGCGUGCAUGGCUTT-3′ and antisense 5′-AGCCAUGCACGCUCGAAGUTT-3′; siRNA-V1G1, sense 5′-AGAAGAAGCUCAGGCUGAATT-3′ and antisense 5′-UUCTGCCTGAGCUUCUUCUTT-3′;
For silencing, HeLa cells were transfected using Oligofectamine Transfection Reagent (Invitrogen) according to the manufacturer’s instructions for 72 h, re-plated and left 48 h before performing further experiments. MCF7 cells were transfected using Metafectene SI (Biontex) according to the manufacturer’s instructions and were processed 48 h after the transfection.
The pCDNA3_2xHA was generated by cloning the fragment of 2xHA 5′ TAC CCA TAC GAT GTT CCG GAT TAC GCT TAC CCA TAC GAT GTT CCG GAT TAC GCT 3′, flanked by a sequence containing the site for KpnI enzyme, into the vector pCDNA3 (Invitrogen).
MCF7 and HeLa cells were silenced with small interfering RNAs (siRNAs) purchased from Eurofins Genomics (Ebersberg, Germany). We used the following oligonucleotides: control RNA, sense 5′-ACUUCGAGCGUGCAUGGCUTT-3′ and antisense 5′-AGCCAUGCACGCUCGAAGUTT-3′; siRNA-V1G1, sense 5′-AGAAGAAGCUCAGGCUGAATT-3′ and antisense 5′-UUCTGCCTGAGCUUCUUCUTT-3′;
For silencing, HeLa cells were transfected using Oligofectamine Transfection Reagent (Invitrogen) according to the manufacturer’s instructions for 72 h, re-plated and left 48 h before performing further experiments. MCF7 cells were transfected using Metafectene SI (Biontex) according to the manufacturer’s instructions and were processed 48 h after the transfection.
10
Lentiviral Transduction and Stable Cell Line Generation
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The appropriate lentivirus packaging kit was purchased from Invitrogen (Lipofectamine™ LTX Reagent with PLUS™ Reagent, REF: 15338100) and used according to the manufacturer’s guidelines. Harvested viral particles (lenti-Vector, lenti-GSDMD-NT, lenti-eGFP) were added to TC-1, 4T1 or CT26 cells. After 24 hours, the cells were washed with RPMI 1640 media and puromycin (5 μg/mL) was added to screen for stably expressed cell lines. The selected cell lines were maintained with puromycin at a concentration of 2.5 μg/mL
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