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Plv azurite

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The PLV-Azurite is a plasmid vector that expresses the Azurite fluorescent protein. Azurite is a blue fluorescent protein derived from the Cnidarian Anthozoa.

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Lab products found in correlation

Pmd2 g, by Addgene (3 mentions) Trypsin edta, by Thermo Fisher Scientific (3 mentions) Plv egfp, by Addgene (2 mentions) Lego c2, by Addgene (2 mentions) Neuraminidase, by Merck Group (1 mentions) Pcmvr8, by Addgene (1 mentions) Pdonr223 erbb2, by Addgene (1 mentions) Pinducer21, by Addgene (1 mentions) Gateway lr clonase kit, by Thermo Fisher Scientific (1 mentions) Plv gfp, by Addgene (1 mentions) Fluostar omega fluorescent plate reader, by BMG Labtech (1 mentions) Fugene hd transfection reagent, by Promega (1 mentions) Egm 2 singlequot, by Lonza (1 mentions) Ccd 19lu, by American Type Culture Collection (1 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions) Egm 2, by Lonza (1 mentions) Lego v2, by Addgene (1 mentions) Dulbecco modified eagle medium (dmem), by Corning (1 mentions) Fetal bovine serum (fbs), by Gemini Bio (1 mentions) Egm 2 medium, by Lonza (1 mentions)

8 protocols using plv azurite

1

Engineered HER2 Expression in Primary Cells

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An inducible HER2 expression vector was constructed by subcloning the ERBB2 open reading frame from pDONR223-ERBB2 (23888; Addgene, Cambridge, MA, USA) into pINDUCER21 (46948; Addgene) using the Gateway LR Clonase kit (Thermo Fisher Scientific) following the manufacturer’s guidelines. Lentiviral particles were generated by co-transfecting HEK293T cells with the packaging plasmids pMD2.G (12259; Addgene) and pCMVR8.2 (12263; Addgene) and either pLV-GFP (36083; Addgene), pLV-Azurite (36086; Addgene) or pINDUCER21-ERBB2 using FuGENE HD transfection reagent (Promega, Madison, WI, USA) following the manufacturer’s guidelines. Virus-containing supernatant was collected 48 h post-transfection.
When primary myoepithelial and luminal cells were infected with lentiviral particles, particles were treated with 20 mU/ml neuraminidase (Sigma-Aldrich) at 37 °C for 30 minutes prior to their addition to cells. Particles provided with SMARTchoice promoter selection plates (GE Dharmacon, Lafayette, CO, USA) were also treated with 20 mU/ml neuraminidase prior to their application to cells; fluorescence intensity values were acquired using a FLUOstar Omega fluorescent plate reader (BMG Labtech, Cary, NC, USA).
Carter E.P., Gopsill J.A., Gomm J.J., Jones J.L, & Grose R.P. (2017). A 3D in vitro model of the human breast duct: a method to unravel myoepithelial-luminal interactions in the progression of breast cancer. Breast Cancer Research : BCR, 19, 50.
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2

Characterization of HUVEC Cells Expressing TIE2 Variants

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HUVEC and retrovirally-transfected HUVEC expressing full-length TIE2-WT or TIE2-L914F were a gift from Dr. Lauri Eklund and Miikka Vikkula, and were previously described20 (link). All the experiments with human cells were performed in accordance with the Institutional Biosafety Committee guidelines and were approved by the Cincinnati Children’s Hospital Medical Center (CCHMC).
Cells were expanded in culture on 1%(w/v) gelatin/PBS-coated plates and Endothelial Cell Growth Medium (EGM-2) (Lonza) supplemented with growth factors (EGM-2 Single Quot, Lonza) and 10% fetal bovine serum (FBS) (HyClone). Human lung fibroblasts (CCD-19Lu) were purchased from ATCC and cultured in DMEM (Invitrogen)/10%FBS. Human retinal pericytes were purchased from Cell Systems and cultured in DMEM (Invitrogen)/10%FBS. GFP (green fluorescent protein) and BFP (blue fluorescent protein) expressing cells were obtained upon infection with lentivirus expressing pLV-eGFP and pLV-Azurite, respectively (Addgene). Pure eGFP or BFP -expressing populations were obtained upon cell sorting (FACS Core at CCHMC). Doxycyclin inducible HUVEC-TIE2-L914F were obtained upon infection of HUVEC with a lentivirus expressing pInducer21-TIE2-L914F (Addgene).
Cai Y., Schrenk S., Goines J., Davis G.E, & Boscolo E. (2019). Constitutive Active Mutant TIE2 Induces Enlarged Vascular Lumen Formation with Loss of Apico-basal Polarity and Pericyte Recruitment. Scientific Reports, 9, 12352.
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3

Isolation and Culture of ECFC-EC

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Human endothelial colony-forming cell-derived endothelial cells (ECFC-EC) are isolated from cord blood with IRB approval according to an established protocol.41 (link) After selection for the CD31+ cell population, ECFC-EC are expanded on gelatin-coated flasks and cultured in EGM2 medium (Lonza). ECFC-EC are used between passages 4–8. Normal human lung fibroblasts (NHLF) are purchased from Lonza and used between passages 6–10. HCT116 colorectal cancer cells were donated from the UC Irvine Chao Family Comprehensive Cancer Center and SW480 colorectal cancer cells were clonally derived and provided by Dr. Marian Waterman. The ECFC-EC and cancer cells were transduced with lentivirus expressing mCherry (LeGO-C2, plasmid # 27339), green fluorescent protein (GFP) (LeGO-V2, plasmid # 27340), or azurite (pLV-azurite, plasmid # 36086) (Addgene, Cambridge, Massachusetts).42,43 (link) Cancer cells and fibroblasts are cultured in DMEM (Corning) containing 10% FBS (Gemini Bio). All cells are cultured at 37 °C, 20% O2, and 5% CO2.
Hachey S.J., Movsesyan S., Nguyen Q.H., Burton-Sojo G., Tankazyan A., Wu J., Hoang T., Zhao D., Wang S., Hatch M.M., Celaya E., Gomez S., Chen G.T., Davis R.T., Nee K., Pervolarakis N., Lawson D.A., Kessenbrock K., Lee A.P., Lowengrub J., Waterman M.L, & Hughes C.C. (2021). An in vitro vascularized micro-tumor model of human colorectal cancer recapitulates in vivo responses to standard-of-care therapy. Lab on a Chip, 21(7), 1333-1351.
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4

Lentiviral CRISPR Plasmid Construction

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LentiCRISPR v2 was a gift from Feng Zhang (Addgene plasmid #52961). pCW57-GFP-2A-MCS was a gift from Adam Karpf (Addgene plasmid #71783). pLV-azurite was a gift from Pantelis Tsoulfas (Addgene plasmid #36086). pET29b was obtained from Novagen. pCS470 tfLC3, pCS625 BFP, psPAX, and pVSV were gifts from Christopher Shoemaker. pGEX-6P1 GST-SGTA was a gift from Robert Keenan. pGEX GST-GET4, pACYC His-cBAG6[1004–1132], pRSETA His-UBL4A, pET28a zebrafish His-ASNA1 and PURExpress 3HA-SEC61B were gifts from Susan Shao. The vector for SEC61B-3F4 was a gift from Ramanujan Hegde.
Morgens D.W., Chan C., Kane A.J., Weir N.R., Li A., Dubreuil M.M., Tsui C.K., Hess G.T., Lavertu A., Han K., Polyakov N., Zhou J., Handy E.L., Alabi P., Dombroski A., Yao D., Altman R.B., Sello J.K., Denic V, & Bassik M.C. (2019). Retro-2 protects cells from ricin toxicity by inhibiting ASNA1-mediated ER targeting and insertion of tail-anchored proteins. eLife, 8, e48434.
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5

Lentiviral Transduction and 3D Tumor Spheroid Co-Culture

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pLV Azurite and pLV Cherry constructs were purchased from Addgene. psPAX2, pMD2.G, and the vector constructs were transfected into 293T cells using a standard calcium phosphate method. Lentivirus particles were collected from the filtered conditioned medium using the Lenti-X-concentrator (Clonetech). To generate fluorescence expressing cell lines, tumour cells or CAFs were infected with lentivirus in DMEM plus polybrene.
For 2D tumour spheroid co-culture assays, D4MAzurite and mCAFsmCherry were cultured to 70% confluence. D4MAzurite cells (1.5 × 105 cells/ml) were seeded in 20 µl hanging drops containing 5% methylcellulose on the inverted lid of the 10 mm petri dish containing PBS. The dishes were incubated for 24 h to allow aggregation into the spheroid. Generation of tumour spheroids was confirmed under light microscopy. mCAFsmCherry were seeded at a 1.5 × 105 cells/ml in six-well plates. Scriptaid treated (7 days) mCAFsmCherry were extensively washed to remove any residual Scriptaid. Tumour spheroids were then collected and transferred to six-well plates previously seeded with control or Scriptaid-treated mCAFsmCherry. The cultures were placed at 37 °C in a humidified incubator under a 5% CO2 atmosphere for 48 h. Images were analysed using NIS Elements software (Nikon).
Kim D.J., Dunleavey J.M., Xiao L., Ollila D.W., Troester M.A., Otey C.A., Li W., Barker T.H, & Dudley A.C. (2018). Suppression of TGFβ-mediated conversion of endothelial cells and fibroblasts into cancer associated (myo)fibroblasts via HDAC inhibition. British Journal of Cancer, 118(10), 1359-1368.
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6

Fluorescent Tagging of Cell Lines

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The cell lines used in this paper are HFL1 (ATCC CCL-153), EA.hy926 (ATCCRL-2922), A549 (ATCC CCL-185), and Lenti-X™ 293T (632180, Takara Bio, CA, USA). These cells were maintained in 2D culture using the complete 2D growth media. 0.25% trypsin-EDTA (90057, Thermo Fisher Scientific, MA, USA) was used during subculture and split ratios were kept being within the suggested range by the manufacturer. Subculture was done every 2–3 days.
Fluorescent tags were introduced using transfer plasmids that were kindly gifted by Pantelis Tsoulfas via Addgene. Specifically, we used pLV-mCherry (36084, Addgene, MA, USA), pLV-Azurite (36086, Addgene, MA, USA), or pLV-eGFP (36083, Addgene, MA, USA) with second-generation lentiviral vectors pMD2.G (12259, Addgene, MA, USA) and psPax2 (12260, Addgene, MA, USA) which were gifted by Didier Trono through Addgene. Viral gene vectors were transfected using PEI 25 K (23966, Polysciences, PA, USA) into Lenti-X™ 293T cells. Lentiviral particles for eGFP, Azurite, and mCherry gene insertions were collected with the media and were transduced into A549, EAhy or HFL1, respectively using polybrene (TR-1003-G, Sigma-Aldrich, MO, USA) for 72 h. Cell purification was then done using fluorescence-activated cell sorting (FACS).
Valdoz J.C., Franks N.A., Cribbs C.G., Jacobs D.J., Dodson E.L., Knight C.J., Poulson P.D., Garfield S.R., Johnson B.C., Hemeyer B.M., Sudo M.T., Saunooke J.A., Kartchner B.C., Saxton A., Vallecillo-Zuniga M.L., Santos M., Chamberlain B., Christensen K.A., Nordin G.P., Narayanan A.S., Raghu G, & Van Ry P.M. (2022). Soluble ECM promotes organotypic formation in lung alveolar model. Biomaterials, 283, 121464.
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7

Characterization of Endothelial Progenitor Cells

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Samples of de-identified human primary endothelial progenitor cells (EPC) and primary human umbilical vein endothelial cells (HUVEC) were obtained from the Hughes Lab (University of California-Irvine). Five EPC lines (EPC-N, EPC-AD, EPC-D8, EPC-10, EPC-BG) and 2 HUVEC lines (HUVEC-216, HUVEC 155) were tested in this study. These cells were isolated from cord blood or umbilical cords with IRB approval, selected for the CD31+ cell population, and expanded on gelatin-coated flasks in EGM-2 medium (CC-3162; Lonza, Walkersville, MD). All cells were used between passages 5–8. Normal human lung fibroblasts (NHLFs, CC-2515) were obtained from Lonza. Colorectal cancer cell line (HCT116) were donated from the UC Irvine Chao Family Comprehensive Cancer Center. Both NHLF and HCT116 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, 10-013-CV; Corning, Corning, NY) supplemented with 10% fetal bovine serum (FBS, 97068–091; VWR, Radnor, PA) and 5 μg/mL gentamicin (15710064; Gibco, Waltham, MA). Endothelial and HCT116 cells were transduced with lentivirus expressing mCherry (LeGO-C2, plasmid #27339; Addgene, Cambridge, MA) or Azurite (pLV-Azurite, plasmid #36086; Addgene).
Liu Y., Sakolish C., Chen Z., Phan D.T., Bender R.H., Hughes C.C, & Rusyn I. (2020). Human In Vitro Vascularized Micro-Organ and Micro-Tumor Models are Reproducible Organ-on-a-Chip Platforms for Studies of Anticancer Drugs. Toxicology, 445, 152601.
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8

Azurite Fluorescent Protein Lentiviral Transduction

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Azurite fluorescent protein lentiviral plasmid was generated by transducing HEK293T using three helper plasmid vectors (pMDLg-pRRE (Plasmid #12251), pRSV-Rev (Plasmid #12253), pMD2.G (Plasmid #12259), all Addgene; Cambridge, MA) and a third-generation lentiviral vector (Addgene pLV-Azurite, Plasmid #36086) in accordance with Washington University protocol 2947. Plasmid titers were then applied to ECs cultured in EGM-2 media with polybrene [5ug/ml] for 24–48 hours, yielding a ~60% stable transfection rate as confirmed by fluorescent microscopy.
Seiler K.M., Bajinting A., Alvarado D.M., Traore M.A., Binkley M.M., Goo W.H., Lanik W.E., Ou J., Ismail U., Iticovici M., King C.R., VanDussen K.L., Swietlicki E.A., Gazit V., Guo J., Luke C.J., Stappenbeck T., Ciorba M.A., George S.C., Meacham J.M., Rubin D.C., Good M, & Warner B.W. (2020). Patient-derived small intestinal myofibroblasts direct perfused, physiologically responsive capillary development in a microfluidic Gut-on-a-Chip Model. Scientific Reports, 10, 3842.
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