Retronectin coated plate

Manufactured by Takara Bio

Sourced in Japan, United States

Retronectin-coated plates are a type of cell culture plate designed to facilitate cell attachment and growth. The plates have a surface coated with the recombinant human fibronectin fragment Retronectin, which promotes cell adhesion and proliferation.

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RetroNectin (590 citations) RetroNectin-coated (40 citations) Retronectin coated 6 well plates (4 citations) RetroNectin-coated (10 μg/mL) 6-well plates (2 citations)

84 protocols using retronectin coated plate

Transduction of Human and Mouse T Cells

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Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors. PBMCs were activated with 50 ng/mL OKT-3 antibody (MACS) and 100 IU/mL IL-2 for two days prior to transduction and maintained in 100 IU/mL thereafter. Trandsuction was performed by centrifugation of activated T cells in media from retroviral producers at 2000× g at room temperature for 1 h on RetroNectin-coated plates (Takara Bio) for two consecutive days. Experiments were performed in compliance with all relevant ethical regulations and in accordance with MSK IRB Protocol 00009377.
Mouse T cells were engineered as previously described^{16 (link)}. Briefly, T-cells were isolated from spleens of naive mice by mechanical disruption using a 100 μm cell strainer. Splenocytes were collected and red blood cells were lysed with ACK (ammonium-chloride-potassium) lysing buffer (ThermoFisher A1049201). Splenocytes were activated overnight with CD3/CD28 Dynabeads (Life Technologies) and 50 IU/mL human IL-2. Activated T cells were transduced by centrifugation with retroviral supernatant from transduced Phoenix-Eco cells on RetroNectin-coated plates (TakaraBio) for 2 consecutive days.

Gardner T.J., Lee J.P., Bourne C.M., Wijewarnasuriya D., Kinarivala N., Kurtz K.G., Corless B.C., Dacek M.M., Chang A.Y., Mo G., Nguyen K.M., Brentjens R.J., Tan D.S, & Scheinberg D.A. (2021). Engineering CAR-T Cells to Activate Small-Molecule Drugs in situ. Nature chemical biology, 18(2), 216-225.

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Retroviral Transduction of Activated T Cells

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Buffy coats from healthy donors were obtained through the Gulf Coast Regional Blood Center, Houston, TX, USA. Peripheral blood mononuclear cells (PBMCs) isolated with Lymphoprep density separation (Fresenius Kabi Norge) were activated using 1 μg/mL anti-CD3 (Miltenyi Biotec) and 1 μg/mL anti-CD28 (BD Biosciences) mAb-coated plates. On day 3, T lymphocytes were transduced with retroviral supernatants using retronectin-coated plates (Takara Bio, Shiga, Japan). After removal from retronectin plates, T cells were expanded in complete medium (45% RPMI 1640 and 45% Click’s medium [Irvine Scientific], 10% FBS [Hyclone], 2 mM GlutaMAX, 100 U/mL penicillin, and 100 μg/mL streptomycin) with IL-7 (10 ng/mL; PeproTech) and IL-15 (5 ng/mL; PeproTech), changing medium every 2–3 days. On days 12–14, cells were collected for in vitro co-culture experiments.

Krokhotin A., Du H., Hirabayashi K., Popov K., Kurokawa T., Wan X., Ferrone S., Dotti G, & Dokholyan N.V. (2019). Computationally Guided Design of Single-Chain Variable Fragment Improves Specificity of Chimeric Antigen Receptors. Molecular Therapy Oncolytics, 15, 30-37.

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Activation and Transduction of Human and Mouse T Cells

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Human T cells were activated and transduced as described previously ²⁵. Briefly, peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor peripheral blood or leukopacks (New York Blood Center, New York, NY, USA). All experiments were performed in compliance with all relevant ethical regulations and in accordance with IRB 095091. PBMCs were activated with 2 μg/ml phytohemagglutinin and 100 IU/ml of IL-2 for 2 days prior to transduction. Mouse T cells were mechanically isolated from spleens and activated using IL-2 and 4 μg/ml of concanavalin A (Millipore Sigma, St. Louis, MO). Transduction was achieved by centrifugation of activated PBMCs and retroviral supernatant on RetroNectin-coated plates on 3 consecutive days (TakaraBio, Kusatsu, Shiga, Japan).

Rafiq S., Yeku O.O., Jackson H.J., Purdon T.J., van Leeuwen D.G., Drakes D.J., Song M., Miele M.M., Li Z., Wang P., Yan S., Xiang J., Ma X., Seshan V.E., Henderickson R.C., Liu C, & Brentjens R.J. (2018). Targeted delivery of a PD-1-blocking scFv by CAR-T cells enhances anti-tumor efficacy in vivo. Nature biotechnology, 36(9), 847-856.

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Retroviral Transduction of Mouse T Cells

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Human embryonic kidney cells (HEK293T) were retrovirally transfected using Fugene transfection reagent following the manufacturer’s protocols (Promega, Madison). Lymph nodes from 6‐ to 8‐week‐old C57BL/6 mice were processed to a single‐cell suspension, and CD4⁺‐positive or CD8⁺‐positive EasySep mouse isolation kits (Stemcell Technologies, Vancouver) were used, according to the manufacturer’s protocol. The purified T cells were stimulated with murine α‐CD3/α‐CD28 Dynabeads (Life Technologies, Carlsbad) at a bead‐to‐cell ratio of 1:1 for 24 h, in T cell media containing 100 IU recombinant human interleukin (IL)‐2 (rhIL‐2) (PeproTech, Rocky Hill). Spinoculation of T cells was performed at 24 and 48 h post‐activation using retrovirus from 293T cells and retronectin‐coated plates (Takara Bio, Kusatsu). Transduced T cells were maintained in T cell media containing 100 IU rhIL‐2.

Abbott R.C., Verdon D.J., Gracey F.M., Hughes‐Parry H.E., Iliopoulos M., Watson K.A., Mulazzani M., Luong K., D’Arcy C., Sullivan L.C., Kiefel B.R., Cross R.S, & Jenkins M.R. (2021). Novel high‐affinity EGFRvIII‐specific chimeric antigen receptor T cells effectively eliminate human glioblastoma. Clinical & Translational Immunology, 10(5), e1283.

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Lentiviral Transduction of T and B Cells

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Freshly isolated unstimulated T and B lymphocytes were seeded in RPMI 1640 medium (Gibco Invitrogen, Auckland, New Zealand) supplemented with 10% FSC (Lonza, Verviers, Belgium) and penicillin/streptomycin (Gibco, Invitrogen, Auckland, New Zealand). T cells were precultivated for 3 days with rIL-7 (20 ng/mL; BD Biosciences), while B cells were stimulated for 24 h with Pansorbin A (Sigma) and IL2 (100 ng/mL; Miltenyi Biotec) before transduction.
5 × 10⁴ T or B cells were seeded in 48-well plates in 100 μL of medium. The volume of concentrated vector for the indicated MOI was preincubated with Vectofusin-1 in serum-free Opti-MEM medium for 10 min. After the incubation period, RPMI/10% FCS medium was added to the mixture to reach a volume of 100 μL and then added on the cells (final concentration Vectofusin = 12 μg/mL). The cells were washed the next day and were replenished with fresh medium and cytokines every 3 days. Three and 6 days after transduction, the percentage of GFP⁺ cells was determined by flow cytometry. Where indicated, transductions were performed on Retronectin-coated plates according to the manufacturer's instructions (Takara Bio).

Radek C., Bernadin O., Drechsel K., Cordes N., Pfeifer R., Sträßer P., Mormin M., Gutierrez-Guerrero A., Cosset F.L., Kaiser A.D., Schaser T., Galy A., Verhoeyen E, & Johnston I.C. (2019). Vectofusin-1 Improves Transduction of Primary Human Cells with Diverse Retroviral and Lentiviral Pseudotypes, Enabling Robust, Automated Closed-System Manufacturing. Human Gene Therapy, 30(12), 1477-1493.

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Isolation and Transduction of Murine HSCs

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Bone marrow cells were isolated and cultured as previously described^{28 (link)}. In short, femurs, tibias, hips, and spines were isolated from donor mice, crushed, and lysed with Ammonium-Chloride-Potassium (ACK) lysing buffer (Gibco Cat# A1049201). LSK cells (lineage^– Sca-1⁺ Kit⁺) were enriched with a CD117 MACS isolation kit (Miltenyi Cat# 130-091-224) and then sorted to purity. The LSK cells were spin transduced with lentiviral vectors on Retronectin-coated plates (Takara Bio Cat# T100B) at a multiplicity of infection (MOI) of 25. LSK cells were then transferred intravenously into irradiated CD45.2⁺ wild-type recipients.

LaFleur M.W., Nguyen T.H., Coxe M.A., Miller B.C., Yates K.B., Gillis J.E., Sen D.R., Gaudiano E.F., Abosy R.A., Freeman G.J., Haining W.N, & Sharpe A.H. (2019). PTPN2 regulates the generation of exhausted CD8+ T cell subpopulations and restrains tumor immunity. Nature immunology, 20(10), 1335-1347.

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Isolation and Transduction of Murine HSCs

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Retroviral Transduction of Activated T Cells

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Buffy coats from healthy donors were purchased from the Gulf Coast Regional Blood Center, Houston, TX. Peripheral blood mono-nuclear cells (PBMCs) isolated with Lymphoprep density separation (Fresenius Kabi Norge) were activated on plates coated with 1 μg/mL CD3 (Miltenyi Biotec) and 1 μg/mL CD28 (BD Biosciences) agonistic mAbs. On day 2, T lymphocytes were transduced with retroviral supernatants using retronectin-coated plates (Takara Bio Inc., Shiga, Japan). Briefly, non-tissue culture treated 24-well plates are coated overnight with 7 mg/mL retronectin in the cold room, washed once with 1 mL medium, coated with 1 mL of the retroviral supernatant per well and centrifuged at 2000 g for 90 min. After removal of the supernatant, 5 × 10⁵ activated T cells are plated, and centrifuged at 1000 g for 10 min. Three days later, T cells are collected and expanded in complete medium (45% RPMI-1640 and 45% Click’s medium (Irvine Scientific), 10% FBS (Hyclone), 2 mM GlutaMAX, 100 unit/mL of Penicillin and 100 mg/mL of streptomycin) with IL-7 (10 ng/mL; PeproTech) and IL-15 (5 ng/mL; PeproTech), changing medium every 2–3 days (Xu et al., 2014 (link)). On day 12–14, cells are collected for in vitro and in vivo experiments. T cells were cultured in IL-7/IL-15 depleted medium for two days prior to being used in in vitro functional assays.

Du H., Hirabayashi K., Ahn S., Kren N.P., Montgomery S.A., Wang X., Tiruthani K., Mirlekar B., Michaud D., Greene K., Herrera S.G., Xu Y., Sun C., Chen Y., Ma X., Ferrone C.R., Pylayeva-Gupta Y., Yeh J.J., Liu R., Savoldo B., Ferrone S, & Dotti G. (2019). Antitumor Responses in the Absence of Toxicity in Solid Tumors by Targeting B7-H3 via Chimeric Antigen Receptor T Cells. Cancer cell, 35(2), 221-237.e8.

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Generation of Bicistronic Retroviral Vectors for HPSE and CAR-GD2 Co-expression

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HPSE cDNA (accession number NM-006665) was cloned into the SFG retroviral backbone that also encodes the GFP [SFG.HPSE(I)GFP] (Supplementary Fig. 12). The construct for the GD2-specific CAR containing the CD28, OX40 and ζ endodomains was previously described (SFG.CAR)^{34 (link)}. We then generated a bicistronic vector to co-express the HPSE and CAR-GD2 using an IRES [SFG.CAR(I)HPSE] (Supplementary Fig. 12). The retroviral vector encoding the fusion protein GFP-firefly luciferase (GFP.FFLuc) for in vivo imaging of T cells and CD19-specific and CSPG4-specific CARs were previously described^{35 (link)}. Transient retroviral supernatant was produced as previously described^{35 (link)}. A specific inhibitor of HPSE, Roneparstat (SST0001) (a chemically modified heparin ¹⁰⁰Na,Ro-H, property of Sigma-tau Research Switzerland S.A.) (3 μg ml)^{13 (link), 36 (link), 37 (link)}, was added to the media during the virus preparation to increase its titer. Activated T lymphocytes were then transduced with retroviral supernatants using retronectin-coated plates (Takara Bio Inc, Shiga, Japan). After removal from the retronectin plates, T-cell lines were maintained in complete T-cell medium in a humidified atmosphere containing 5% CO₂ at 37°C in the presence of IL-2 (50 U mL) for 2 weeks.

Caruana I., Savoldo B., Hoyos V., Weber G., Liu H., Kim E.S., Ittmann M.M., Marchetti D, & Dotti G. (2015). Heparanase promotes tumor infiltration and antitumor activity of CAR-redirected T-lymphocytes. Nature medicine, 21(5), 524-529.

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Genetic Modification of γδ-T Cells for Immunotherapy

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Retroviral and lentiviral vectors were generated to stably transduce K562, an immortalised leukaemia cell line. The retroviral vector was designed to encode the cDNA of CD86, 41BBL and the iC9 suicide gene using SFG retroviral backbone (25 (link)), while the lentiviral vectors encode the cDNA for the CMV-pp65 with or without the costimulatory molecule CD40L. An additional retroviral vector encoding eGFP-Firefly-Luciferase (eGFP-FFLuc) was used to label tumour cells for in-vitro and in-vivo studies, as previously described (26 (link), 27 (link)). For gene-modification, 1 × 10⁶ expanded γδ-T cells/well in a non-tissue culture treated 24 well plate were transduced 4–5 days after II° restimulation using retronectin-coated plates (1 μg/well, Takara Bio, Shiga, Japan) with 1 mL/well of a retroviral vector encoding for a third-generation chimeric antigen receptor (CAR) (28 (link)) specific for GD2 (CAR-GD2.CD28.4-1BBζ) (29 (link)) (Supplementary Figures 1B,C). After removal from the retronectin-coated plates, transduced γδ-T cells were stimulated with aAPCs in bioreactors in the presence of IL2 and IL15 as described below. The transduction efficiency was determined by anti-CAR idiotype staining (1A7) as previously described (30 (link)).

Polito V.A., Cristantielli R., Weber G., Del Bufalo F., Belardinilli T., Arnone C.M., Petretto A., Antonucci L., Giorda E., Tumino N., Pitisci A., De Angelis B., Quintarelli C., Locatelli F, & Caruana I. (2019). Universal Ready-to-Use Immunotherapeutic Approach for the Treatment of Cancer: Expanded and Activated Polyclonal γδ Memory T Cells. Frontiers in Immunology, 10, 2717.

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