The cell line was generated using MACS separation (Miltenyi biotec, autoMACS Pro Separator, #130-092-545, autoMACS Columns #130-021-101) of PDGFRb+ cells that were isolated from the healthy part of the kidney cortex after nephrectomy as previously described in ref. 43 (link). The following antibodies were used for staining the cells and MACS procedure: PDGFRb (R&D #MAB1263 antibody, dilution 1:100) and anti-mouse IgG1-MicroBeads solution (Miltenyi, #130-047-102). The cells were cultured in DMEM media (Thermo Fisher #31885) added 10% FCS and 1% penicillin/streptomycin for 14 days. For immortalization (SV40-LT and HTERT) the retroviral particles were produced by transient transfection of HEK293T cells using TransIT-LT (Mirus). Amphotropic particles were generated by co-transfection of plasmids pBABE-puro-SV40-LT (Addgene #13970) or xlox-dNGFR-TERT (Addgene #69805) in combination with a packaging plasmid pUMVC (Addgene #8449) and a pseudotyping plasmid pMD2.G (Addgene #12259) respectively. Using Retro-X concentrator (Clontech) 48 h post-transfection the particles were concentrated. For transduction, the target cells were incubated with serial dilutions of the retroviral supernatant (1:1 mix of concentrated particles containing SV40-LT or rather hTERT) for 48 h. At 72 h after transfection, the infected PDGFRb+ cells were selected with 2 μg/ml puromycin for 7 days.
Pbabe puro sv40 lt
Manufactured by Addgene
Sourced in United States
PBABE-puro SV40 LT is a plasmid vector used for the expression of the SV40 large T antigen in mammalian cells. The vector contains a puromycin resistance gene for selection of transfected cells.
Automatically generated - may contain errors
Lab products found in correlation
Pumvc, by Addgene (4 mentions)
Pmd2 g, by Addgene (3 mentions)
Retro x concentrator, by Takara Bio (2 mentions)
Xlox dngfr tert, by Addgene (2 mentions)
Pcmv vsv g, by Addgene (2 mentions)
Pbabe hygro htert, by Addgene (2 mentions)
Glutamax medium, by Thermo Fisher Scientific (2 mentions)
D5796, by Merck Group (2 mentions)
Pmxs blast mcgas ha, by InvivoGen (2 mentions)
Puno1 mcgas ha3x, by InvivoGen (2 mentions)
Pcl eco, by Addgene (2 mentions)
Dmem media, by Thermo Fisher Scientific (1 mentions)
Anti mouse igg1 microbeads solution, by Miltenyi Biotec (1 mentions)
Automacs pro separator, by Miltenyi Biotec (1 mentions)
Hygromycin, by InvivoGen (1 mentions)
Puromycin selection, by Santa Cruz Biotechnology (1 mentions)
Ppackh1 hiv lentivector packaging kit, by System Biosciences (1 mentions)
Countess, by Thermo Fisher Scientific (1 mentions)
Quikchange 2 site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Pspcas9 bb 2a puro, by Addgene (1 mentions)
16 protocols using pbabe puro sv40 lt
1
Immortalized PDGFRb+ Kidney Cells
2
Establishment of Immortalized Human Dermal Fibroblasts
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MuLV retroviral particles were obtained using HEK 293T as the packaging cell line and pBABE-puro-SV40LT (gift from Thomas Roberts; Addgene plasmid #13970) [50 (link)] and pBabe-hygro-hTERT, pUMVC and pCMV-VSV-G (gifts from Bob Weinberg; Addgene plasmids #1773, #8449 and #8454 respectively) [51 (link), 52 (link)] in accordance with the protocol of Scott Dessain and Hua-yin Yu, Weinberg Lab (https://www.addgene.org/static/data/84/58/16578800-af64-11e0-90fe-003048dd6500.pdf ). LV pmiRZip AS miR-155, LV pmiRZip CTRL, LV pCDH_miR-155 and LV pCDH_CTRL were obtained by using the pPACKH1 HIV Lentivector Packaging Kit (System Biosciences). HDFLT/hTERT LV AS miR-155 and control (LV AS CTRL) cells were developed by sequential infections with polybrene (5 μg/ml). Briefly, normal HDF were infected at passage P4, with RV LTSV40 for 3 hours, then were washed and infected overnight with LV AS miR-155, or LV AS CTRL. After 7 days of puromycin selection (0.5 μg/ml) (Santa Cruz Biotechnology), at P6, cells were infected with RV hTERT for 3 hours and selected with hygromycin (50 μg/ml) (InvivoGen). Since LV AS miR-155 contains the copGFP reporter gene, GFP-positive cells were sorted using the FACSAriaIII, 100 μm nozzle (BD Biosciences).
3
Transformation and Tumor Formation Assays
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Low-passage MEFs were transformed using retrovirus prepared with pBABE-puro SV40 LT and pWZL Blast H-Ras (Addgene, Cambridge, MA, USA). After antibiotic selection, cells were split into six-well plates at 1×105 cells per plate to evaluate cell proliferation. Cell numbers were determined using an automated cell counter (Countess, Invitrogen, Waltham, MA, USA). For soft agar assays, 5×103 cells were suspended in complete medium containing 0.3% Noble agar and seeded into a six-well plate containing 0.6% Noble agar in the wells. Medium was changed twice per week. After 21 days, colonies were stained with 0.005% crystal violet and counted by using a dissecting microscope. For in vivo tumor formation, 5×105 cells were injected subcutaneously into athymic nude mice (Foxn1nu/Foxn1nu; Jackson Laboratory). For syngeneic xenografts, 1×105 cells were injected subcutaneously into WT or miR-301a−/− mice. Tumor size was measured every 3 days. Tumor volume was calculated by the formula of width2×length/2.
4
Generation and Characterization of cGAS MEFs
5
Circadian Rhythm Regulation Constructs
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Bmal1, Tip60 ORFs were amplified by PCR and cloned into pcDNA3.1 vector containing either C-terminal myc- or V5-tags. Single site mutations were introduced by using the QuikChange II Site-directed Mutagenesis Kit (Agilent Technologies). Tip60V5 and Tip60C369A;E403Q;V5 were cloned into pLenti PGK Hygro DEST vector. Bmal1-FLAG was from M.J. Rossner (LMU Munich). Myc-Clock and myc-ClockmutA were from P. Sassone-Corsi. Lentiviral Bmal1-dLuc reporters were from S.A. Kay (USC Los Angeles). pBABE-puro SV40 LT (#13970), pCL-Eco (#12371), pMD2.G (#12259), psPAX2 (# 12260), pLenti PGK Hygro DEST (#19066), and pSpCas9(BB)−2A-Puro (# 48139) plasmids were from Addgene.
6
Isolation and Immortalization of Cardiac Gli1+ Cells
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Gli1CreER;tdTomato mice were sacrificed 2 weeks after tamoxifen treatment (Carbolution, CC99648). Cardiac tdTomato+cells were sorted by FACS and immortalized 14 days later with pBABE-puro SV40 LT (Addgene: #13970; a gift from Thomas Roberts). Retroviral particles were produced by transient co-transfection of HEK293T (ATCC, #CRL3216, Lot:70008735, aliquots from passage 2 were used for the experiments) cells with pCL-Eco (Addgene plasmid #12371, a gift from Inder Verma). In total, 72 h after transduction of Gli1+ cells, infected cells were selected with 7-day puromycin titration.
7
Generation and Characterization of cGAS MEFs
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HeLa cells were purchased by ATCC, maintained in DMEM (D5796, Sigma) supplemented with 10% FBS (97068-085, VWR) and kept at low passage to minimize drift. Primary cGAS−/− MEFs were generated from E13.5 embryos in accordance with animal use protocols approved by the Institutional Animal Care and Use Committee (IACUC) at Texas A&M University, maintained in DMEM supplemented with 10% FBS, then transduced with retroviruses encoding SV40 large T antigen (pBABE-puro SV40 LT, Addgene) plasmid to obtain immortalized cGAS−/− MEF (cGAS−/− MEF SV40I). cGAS−/− MEF SV40I cells were then reconstituted with mouse cGAS construct (pMXs-blast-mcGAS-HA cloned from pUNO1-mcGAS-HA3x, InvivoGen) to generate cGAS−/− MEF SV40I+mcGAS-HA cells. HEK 293T cells (ATCC, CRL-3216) were cultured in DMEM (1×) + GlutaMAX medium (Gibco, 10569-010) supplemented with 10% FBS (Gibco, 26140-095), penicillin (100 U/ml) and streptomycin (100 μg/ml) (Lonza, 17-602E) at 37 °C in a humidified atmosphere with 5% CO2. The cells have been authenticated by validating cGAS, STING, or actin by western blotting or immunofluorescence and have been tested negative for mycoplasma contamination.
8
Oncogene and Activator Cassette Construction
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The oncogene cassette was constructed in multiple cloning steps. The following plasmids were used in the construction: the pLVCT‐rtTR‐KRAB‐2SM2 (Addgene plasmid #11779), and the pBABE‐puro‐SV40LT (Addgene plasmid #13970); the YFP‐F2A‐KRAS (isoform‐B) G12D‐T2A‐cMYC, CAG‐RFP, and the TRE element were ordered from GenScript (Piscataway, NJ, USA). The activator cassette was constructed from the following plasmids: 12.4‐kb murine villin promoter (Addgene plasmid #19358), blue fluorescent protein (BFP) mTurquoise2 (Addgene plasmid #54842) (Goedhart et al., 2012 ), PGK‐FlpO (codon optimized), and Cre‐ERT2 (gifts from Frank Schnütgen, Goethe University, Germany). Both cassettes included left and right inverted repeats (LIR and RIR) and LoxP elements from the floxed‐Ei‐Ubi‐PSEN1M146I plasmid described in Jakobsen et al. (2013 ). The oncogene and activator cassette are available from Addgene (deposited as Addgene plasmid #67277 and #67278, respectively).
9
Generation of Immortalized Coronary SMCs
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To generate immortalized SMC (iSMC) lines, primary human coronary artery SMCs were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Thermo Scientific) with 4.5 g/L glucose, 10% FBS, 1% P/S at 37°C in humidified 5% CO2 (Thermo Fisher) and immortalized using SV40LT and HTERT. Retroviral particles were produced by transient transfection of HEK293T cells using TransIT-LT (Mirus). Two types of amphotropic particles were generated by co-transfection of plasmids pBABE-puro-SV40-LT (Addgene) or xlox-dNGFR-TERT (Addgene) in combination with a packaging plasmid pUMVC (Addgene) and a pseudotyping plasmid pMD2.G (Addgene). Retroviral particles were 100x concentrated using Retro-X concentrator (Clontech) 48 h post-transfection. Cell transduction was initiated by incubating the target cells with the retroviral supernatants for 48 h. After 7 days, the infected cells were selected with 2 μg/ml puromycin for 72 h.
10
Neonatal Rat Cardiomyocyte Isolation and Characterization
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Primary cultures of neonatal rat cardiomyocytes were prepared using standard methods (Zhang et al, 2010 ). Rat H9c2 cardiomyoblasts, and HEK293 and U2OS cells were from ATCC. MEFs were prepared from 13.5‐day‐old embryos of Nox4−/− and littermate WT mice, and immortalized with SV40 large T antigen. Cell transfections with plasmids or siRNA were performed using Lipofectamine or FuGENE reagent (Promega). Plasmid sources were as follows: ATF4 (Addgene #24874), GADD34‐Flag Mouse MyD116.PFLAG.CMV2 (Addgene # 21834), GADD34 Mouse MyD116.delC.pBABEpu (Addgene # 21835), and pBABE‐puro SV40 LT (Addgene # 13970). siRNA sequences are provided in the Appendix . Cell viability was assessed using an MTT assay.
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