Rep pt1

Manufactured by InvivoGen

Rep-pt1 is a plasmid expression vector designed for the production of recombinant proteins in mammalian cells. It features a CMV promoter and a SV40 polyadenylation signal, which facilitate efficient transcription and mRNA stabilization. The vector also includes a neomycin resistance gene for selection of transfected cells.

Automatically generated - may contain errors

Lab products found in correlation

Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions) Sodium pyruvate, by Thermo Fisher Scientific (2 mentions) Mem non essential amino acids solution, by Thermo Fisher Scientific (2 mentions) Accutase, by Thermo Fisher Scientific (2 mentions) Poly l lysine (pll), by Merck Group (2 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Lt07 710, by Lonza (1 mentions) Matrigel basement membrane matrix high concentration, by BD (1 mentions) Dmem high glucose media, by Merck Group (1 mentions) Rpmi 1640 media, by Merck Group (1 mentions) Carbonic anhydrase activity assay kit, by Abcam (1 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions) Phosstop, by Merck Group (1 mentions) Complete protease inhibitor cocktail, by Roche (1 mentions) Penicillin streptomycin solution, by Corning (1 mentions) Ant nr 2, by InvivoGen (1 mentions) Mda mb 231, by Sartorius (1 mentions) Hek293t, by Sartorius (1 mentions) High glucose dmem, by Sartorius (1 mentions)

7 protocols using rep pt1

Orthotopic Implantation of KPC-derived PDAC Cells

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The KPC-derived PDAC cell line (TB32048) previously generated from a female C57BL/6 KPC mouse was provided as a generous gift from David Tuveson laboratory. TB32048 was cultured for 3–4 passages at maximum 80% confluency in 10% fetal calf-serum (#A15-104, GE Healthcare) in DMEM (#E15-810, PAA) + 100 μg/ml penicillin/streptomycin (#P11-G10, PAA) in a T175 flask in standard conditions (37°C, 5% CO₂) and tested regularly in house for mycoplasma (#rep-pt1, Invivogen and #LT07-710, Lonza). Cells were dissociated using 0.1% trypsin (PBS) (#594-18C, Sigma) for 10 min at 37°C and resuspended in PBS and BD Matrigel™ Basement Membrane Matrix High Concentration (#354248, BD Biosciences) in a ratio of 1:1. 1,000 cells in 5 μl was injected into the pancreas using a Hamilton® syringe, 700 series (#10100332, Fisher Scientific). For the experiments used to assess CD138^hi cells and anti-CD20 at day 10, 10,000 cells in 30 μl Matrigel, or Matrigel only sham surgery was performed. The peritoneal wall was sutured using 6/0-gauge coated vicryl sutures™ (#W9500T, Ethicon) and skin closed using two 9 mm Clay Adams Clips (#IN015A, VetTech Solutions) and an Autoclip® applier (#IN015B, VetTech Solutions).

Spear S., Candido J.B., McDermott J.R., Ghirelli C., Maniati E., Beers S.A., Balkwill F.R., Kocher H.M, & Capasso M. (2019). Discrepancies in the Tumor Microenvironment of Spontaneous and Orthotopic Murine Models of Pancreatic Cancer Uncover a New Immunostimulatory Phenotype for B Cells. Frontiers in Immunology, 10, 542.

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CRISPR-edited U-2 OS Cell Line Cultivation

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U-2 OS-CRISPR-NUP96-mEGFP clone #195 (300174, CLS GmbH) and U-2 OS (300364, CLS GmbH) cells²⁸ were obtained in January 2020, and were used without authentication. Cells were grown in McCoy’s 5A (modified) media (Thermo Fisher Scientific, #16600082) supplemented with 100 U/mL penicillin-streptomycin (Thermo Fisher Scientific, #15140148), 1 mM sodium pyruvate (Thermo Fisher Scientific, #11360070), 1X MEM non-essential amino acids solution (Thermo Fisher Scientific, #11140050), and 10% (v/v) fetal bovine serum (Thermo Fisher Scientific, #A3160401) in 5% (v/v) CO₂ enriched air at 37°C. Cells were typically grown to ~95% confluency, were split using Accutase (Thermo Fisher Scientific, #A1110501), and were tested monthly for mycoplasma contamination (InvivoGen, #rep-pt1). For microscopy experiments, freshly split cells were grown overnight (< 3% confluence) on coverslips pre-treated with 0.01% poly-L-lysine (Sigma, #P4832) for 5 min, which reduced cell-detachment after permeabilization.

Chowdhury R., Sau A, & Musser S.M. (2022). Super-resolved 3D Tracking of Cargo Transport Through Nuclear Pore Complexes. Nature cell biology, 24(1), 112-122.

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In Vitro Assays for Mammary Cell Lines

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All cells were maintained at 37°C in humidified incubators with 5% CO2 at atmospheric oxygen levels. 4T1 cells were a gift from Dr. Siyuan Zhang, University of Notre Dame. EpH4 and NMuMG cells were a gift from Dr. Zena Werb, University of California, San Francisco. 4T1 cells were grown in RPMI1640 media (Sigma R6504) with 10% FBS. EpH4 cells were cultured in DMEM High Glucose media (Sigma D5648) with 5% FBS. NMuMG cells were cultured in DMEM High Glucose media with 10% FBS and 1 μg/mL insulin. All cells were routinely tested for mycoplasma by PCR (Genlantis MY01050) and colorimetric detection kit (InvivoGen rep-pt1) according to manufacturer’s directions. Quality control of the cell lines was maintained by continual authentication of morphology and growth rate and were used less than 16 passages. Mouse cell lines were not authenticated genetically.
Detailed information on various in vitro experiments - proliferation, scratch, 3-D organoid and contact inhibition assays, cell size, gene knockdown, dependence, ROS, NO detection, urea and NADP+ levels, glycerol quantification and stress tolerance assays can be found in the Supplementary Methods.

Dai C., Charlestin V., Wang M., Walker Z.T., Miranda-Vergara M.C., Facchine B.A., Wu J., Kaliney W.J., Dovichi N.J., Li J, & Littlepage L.E. (2020). Aquaporin-7 Regulates the Response to Cellular Stress in Breast Cancer. Cancer research, 80(19), 4071-4086.

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Carbonic Anhydrase Activity Assay in HPDE6c7 Cells

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HPDE6c7 cells (RRID:CVCL_0P38) were authenticated by ATCC and tested negative for mycoplasma using a kit from InvivoGen (rep-pt1). HPDE6c7 cells (Furukawa et al., 1996 (link)) were cultured in DMEM (Life Technologies 11995073), 10% FBS (Corning, 35011CV), 1X Penicillin:Streptomycin solution (Corning, 30-002-CI). For cell counting, 25,000 cells were seeded in a sterile six-well TC-treated plate (Corning, 353046). Values depicted for all cell culture experiments represent the average of at least three independent experiments.
For carbonic anhydrase activity assays, cell lysates were prepared using standard protocols and cell lysis buffer (Cell Signaling Technologies, 9803S) containing 100 mM PMSF, 1X cOmplete Protease Inhibitor Cocktail (Roche, 11697498001), and 1X PhosSTOP (Sigma Aldrich, 4906845001). Carbonic anhydrase activity was measured using the carbonic anhydrase activity assay kit (Biovision, K472-100). For normalization, equal amounts of protein (10 µg) per sample were used in the assay. Protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, 23225).

Hendley A.M., Rao A.A., Leonhardt L., Ashe S., Smith J.A., Giacometti S., Peng X.L., Jiang H., Berrios D.I., Pawlak M., Li L.Y., Lee J., Collisson E.A., Anderson M.S., Fragiadakis G.K., Yeh J.J., Ye C.J., Kim G.E., Weaver V.M, & Hebrok M. (2021). Single-cell transcriptome analysis defines heterogeneity of the murine pancreatic ductal tree. eLife, 10, e67776.

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CRISPR-edited U-2 OS Cell Line Cultivation

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Chowdhury R., Sau A, & Musser S.M. (2022). Super-resolved 3D Tracking of Cargo Transport Through Nuclear Pore Complexes. Nature cell biology, 24(1), 112-122.

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Cultivation of Human Cancer Cell Lines

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Human cancer cell lines used at MD Anderson were obtained as follows: EOL1, MONOMAC1, NB4, OCIAML3 (DSMZ, #ACC-386 #ACC-252 #ACC-207 #ACC-582); MOLM13 and NOMO1 (Fisher, #NC0442994 #NC1515509); MV411 (ATCC #CRL-9591). Identities were confirmed upon receipt and prior to experiments by STR typing (MDACC Characterized Cell Line Core). The absence of mycoplasma was confirmed monthly (Invivogen #rep-pt1). All cell lines were grown at 37 °C in 5% CO₂ in low attachment flasks (Greiner) and maintained at less than 1 M cells ml⁻¹. All but one line were cultured in RPMI-1640 with 25 mM HEPES ((Sigma #R5886) supplemented with 10% FBS (Sigma # F0926), 2 mM Glutamax (Gibco #35050061), 1 mM sodium pyruvate (Gibco #11360070), 10,000 units ml⁻¹ penicillin (Gibco #15140122), 10 mg ml⁻¹ streptomycin (Gibco #15140122), and 100 µg ml⁻¹ Normocin (Invivogen #ANTNR2). Complete medium was additionally supplemented with 0.1 mM nonessential amino acids (Gibco #11140050) for MONOMAC1.

Lenoir W.F., Morgado M., DeWeirdt P.C., McLaughlin M., Griffith A.L., Sangree A.K., Feeley M.N., Esmaeili Anvar N., Kim E., Bertolet L.L., Colic M., Dede M., Doench J.G, & Hart T. (2021). Discovery of putative tumor suppressors from CRISPR screens reveals rewired lipid metabolism in acute myeloid leukemia cells. Nature Communications, 12, 6506.

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Cell Line Maintenance and Validation

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Cell lines and cell culture HEK293T and EMT6 cells were purchased from the American Type Culture Collection in 2018, and MDA-MB-231 and MDA-MB-468 cells were purchased from the Cell Bank of the Chinese Academy of Sciences in 2018. HEK293T and MDA-MB-231 cells were maintained in DMEM high glucose (Biological Industries) supplemented with 10% heat-inactivated FBS (Gibco). MDA-MB-468 cells were maintained in RPMI 1640 (Biological Industries) medium supplemented with 10% heat-inactivated FBS, and EMT6 cells were maintained in Waymouth's Medium (Gibco) supplemented with 15% heatinactivated FBS. All cells were maintained in a humidified incubator with 5% CO 2 at 37 C. Cell lines were not authenticated since purchase and were cultured for fewer than 10 passages. Cell lines were routinely tested for Mycoplasma using a Mycoplasma contamination detection kit (rep-pt1, InvivoGen).

Xia L., Zheng Z., Liu J.Y., Chen Y.J., Ding J., Hu G.S., Hu Y.H., Liu S., Luo W.X., Xia N.S, & Liu W. (2021). Targeting Triple-Negative Breast Cancer with Combination Therapy of EGFR CAR T Cells and CDK7 Inhibition. Cancer immunology research, 9(6).

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