HEK293T and Rat-2 cells were maintained in DMEM supplemented with heat-inactivated 10% fetal bovine serum (Invitrogen) and Pen/Strep (HyClone). Luciferase expressing, single cycle of replication competent MLV particles (MLV/luc) were derived via co-transfection of HEK293T cells with pLNC-luc (luciferase expressing retroviral expression vector) and pCL-Eco (MLV packaging vector that expresses MLV Gag, Pol and ecotropic Env glycoproteins; Imgenex) plasmids, as previously described25 (link). For the virus fusion assay described below, HEK293T cells were co-transfected with pLNC-luc, pCL-Eco and S15-BlaM plasmids. To generate the S15-BlaM expression plasmid that expresses a Src-eptiope tagged β-lactamase fusion protein upon transfection, the N-terminal 15 amino acid sequence of c-Src (S15) was cloned in frame upstream of β-lactamase orf in pcDNA3.1/zeo + (Invitrogen) eukaryotic expression plasmid. S15-BlaM is incorporated into MLV particles upon virus particle budding from the plasma membrane of virus-producing cells26 ,27 (link). Virus-containing supernatants were harvested 2 days post transfection, and passed through 0.45 µm filters. Virus was concentrated by ultracentrifugation on a 20% sucrose cushion [28,000 rpm at 4 °C for 2 hours with a SW32 Ti rotor (Beckman Coulter)]. Virus pellets were resuspended in PBS, aliquoted, and stored at −80 °C until further use.
Pcl eco
Manufactured by Novus Biologicals
Sourced in United States
The PCL-Eco is a laboratory instrument designed for the quantitative detection of nucleic acids. It utilizes a luminescent-based detection method to quantify the presence and concentration of specific DNA or RNA sequences in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Pcmv vsv g, by Addgene (5 mentions)
Pcmv dr8.2 dvpr, by Addgene (5 mentions)
293t 17 cells, by American Type Culture Collection (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Pbabe puro ppar gamma2 vector, by Addgene (2 mentions)
Polybrene, by Merck Group (2 mentions)
Concanavalin a (cona), by Merck Group (2 mentions)
2 mercaptoethanol, by Merck Group (2 mentions)
Sodium pyruvate, by Corning (2 mentions)
Pen strep, by Corning (2 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
Non essential amino acids (neaa), by Corning (2 mentions)
Pcdna3.1 zeo, by Thermo Fisher Scientific (1 mentions)
Sw32ti rotor, by Beckman Coulter (1 mentions)
X tremegene 9, by Merck Group (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
Gentra puregene cell kit, by Qiagen (1 mentions)
Facsaria, by BD (1 mentions)
Centricon plus 70 filter, by Merck Group (1 mentions)
17 protocols using pcl eco
1
Production and Characterization of MLV Particles
2
Complex Reprogramming of Mouse Fibroblasts
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MEFs were derived from E13.5 C57BL/6J embryos (the Jackson laboratory: 000664). Retroviral particles were produced by transfecting 293T-17 cells (ATCC: CRL-11268) with the pGCDN-Sam construct containing Hnf4α-t2a-Foxa1/Fos/Yap1, along with packaging construct pCL-Eco (Imgenex). Lentiviral particles were produced with the envelope construct pCMV-VSV-G (Addgene plasmid 8454), the packaging construct pCMV-dR8.2 dvpr (Addgene plasmid 8455), and the shRNA expression vector for the respective candidate TF to be knocked down. For generation of the complex CellTag library, lentiviral particles were produced by transfecting 293T-17 cells (ATCC: CRL-11268) with the pSMAL-CellTag construct, along with packaging constructs pCMV-dR8.2 dvpr (Addgene plasmid 8455) and pCMV-VSVG (Addgene plasmid 8454). For iEP reprogramming, MEFs (< passage 6) were converted to iEPs as in Biddy et al. (2018 (link)), modified from (Sekiya and Suzuki, 2011 (link)). Colony-formation assays were performed as in Biddy et al. (2018 (link)). Perturb-seq was performed as previously described (Adamson et al., 2016 (link)). Single-cell libraries were prepared using the 10x Genomics Chromium platform.
3
Lentiviral and Retroviral Production
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Lentiviruses were produced by transfecting HEK293T cells with lentiviral pSMAL vector and packing plasmids pCMV-dR8.2 dvpr (Addgene plasmid 8455) and pCMV-VSV-G (Addgene plasmid 8454) using X-tremeGENE 9 (Sigma-Aldrich). Viruses were collected 48 and 72 h after transfection. Retroviruses were similarly produced, with retroviral pGCDNSam vector and packaging plasmid pCL-Eco (Imgenex).
4
Retroviral and Lentiviral Transduction of Adipocytes
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pBABE-puro-PPAR gamma2 vector was obtained from Addgene (#8859). Retroviral packaging Bosc-23 cells were cotransfected with the targeting plasmid and packaging vector pCL-eco (Imgenex, Sorrento Valley, CA). Lentiviral particles were produced by transient transfection of 293 T cells with a packaging plasmid psPAX2 and envelope plasmid pMD2.G together with pSin-EF2-Puro vector alone or pSin-EF2-FAM13A-FLAG-Puro. 48 h after transfection, the culture media containing the virus particles were collected and mixed with DMEM 10% FBS at 1:1 to infect 3T3-L1 preadipocytes or NIH-3T3 fibroblasts in the presence of 8 μg/ml polybrene. Cells were then selected with 2 µg/ml puromycin for at least 4 days before analysis or differentiation.
5
Retroviral Transduction of Activated B Cells
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B cells isolated and activated in vitro in the presence of LPS plus IL‐4 were transduced as described in 35 . Briefly, precursor miR‐25‐GFP construct vectors were cloned in pMXPIE‐Btk by PCR amplification of genomic DNA isolated from mouse B cells using the Gentra Puregene cell kit (Qiagen) with specific primers to amplify the precursor mmu‐miR‐25 sequence and its flanking 50 bp genomic sequences obtained from miRBase. Retroviral supernatants were produced by transiently co‐transfecting 293T‐HEK cells with pCL‐Eco (Imgenex) and pre‐miRNA‐GFP retroviral vectors or pMXPIE‐Btk retroviral vector, using calcium phosphate or Lipofectamine 2000 (Thermo Fisher scientific). Mouse primary B cells were then transduced with retroviral supernatants for 20 h in the presence of 8 μg/ml polybrene (Sigma), 10 mM Hepes (Invitrogen), and 50 μM β‐mercaptoethanol (Invitrogen). GFP+ cells were sorted at 48 h post‐transduction (FACSAria, BD Biosciences) and processed for QIAZOL cell lysis, RNA extraction, and qPCR analysis. GFP+ cells were stained with DAPI to detect nuclei, and expression of IgG1, Ki67, and annexin was determined by flow cytometry.
6
Antagonizing miR-24 with shRNA and miRNA inhibitors
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ShRNA antagonist of miR-24 (miArrest miRNA inhibitor) or scrambled non-targeting shRNA was expressed in a lentiviral vector co-expressing mCherry and puromycin resistance (GeneCopoeia, Rockville, MD). For supplementary experiments miR-ZIP vectors to deliver an independent miR-24 targeting shRNA and scrambled shRNA were used (System Biosciences, Mountain View, CA). pLKO.1 lentiviral plasmids expressing Trb3 ShRNA antagonists were obtained from Thermo Scientific (Waltham, MA). pVSV-G, pMDL, and pRSV-REV plasmids (National Gene Vector Biorepository, Indianapolis, IN) were co-transfected with lentiviral plasmid into 293FT cells with Lipofectamine 2000 Transfection Reagent (Invitrogen, San Diego, CA). For retroviral production MigR1-EGFP, MigR1-Trib3, MSCV-EGFP, and MSCV-mirn23a retroviral plasmids were co-transfected into 293FT cells together with the retroviral packaging vector pCL-Eco (Imgenex) using Lipofectamine 2000 (Invitrogen). For both lentivirus and retrovirus production 48h and 72h post-transfection viral supernatants were harvested and concentrated with Centricon Plus-70 filters (Millipore). MigR1-Trib3 plasmid was kindly provided by Dr. Keyong Du, Tufts University, Boston, MA.
7
Retroviral Transduction of T Cells
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Two hundred ninety-three T cells were transfected with a mixture of DNA plasmids containing 1.5 µg of Pcl-Eco (IMGENEX), 2.5 µg of pMIGR1 vector or pMIGR1 harboring Slug by Fugene HD (Roche) according to the manufacturer’s instruction. Virus-containing media were collected 24 h after transfection and filtered through a 0.45-µm pore-size filter. SCs were transduced with retrovirus-containing media supplemented with 4-µg ml−1 polybrene (Sigma), followed by centrifugation at 1000 × g for 1 h.
8
Engineered EGFRvIII CAR T Cell Protocol
9
Lentiviral and Retroviral Transduction
10
Retroviral and Lentiviral Transduction of Adipocytes
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