Retronectin coated

Manufactured by Takara Bio

Sourced in Japan, France

RetroNectin-coated is a laboratory product that serves as a cell culture surface. It is designed to enhance cell attachment and proliferation for various cell-based applications.

Automatically generated - may contain errors

Lab products found in correlation

Interleukin 6 (il 6), by Thermo Fisher Scientific (3 mentions) Packaging plasmids, by Takara Bio (3 mentions) Polybrene, by Merck Group (3 mentions) Nalm6 cells, by Takara Bio (2 mentions) D luciferin, by PerkinElmer (2 mentions) Facsaria 2, by BD (2 mentions) Interleukin 3 (il 3), by Thermo Fisher Scientific (2 mentions) Stemspan serum free expansion medium, by STEMCELL (2 mentions) X vivo 10 media, by Lonza (2 mentions) Fugene, by Promega (2 mentions) Moflo flow sorter, by Beckman Coulter (2 mentions) Megacult, by STEMCELL (2 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions) Thrombopoietin tpo, by R&D Systems (1 mentions) Interleukin 6 (il 6), by R&D Systems (1 mentions) Interleukin 3 (il 3), by R&D Systems (1 mentions) Flt3l, by Thermo Fisher Scientific (1 mentions) T cell enrichment column, by R&D Systems (1 mentions) Untouched cd8 t cell isolation kit, by Miltenyi Biotec (1 mentions)

40 protocols using retronectin coated

Lentiviral Transduction of NALM6 Cells

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Lentivirus including GFP-luciferase reporter genes were produced as previous described ³³. NALM6 cells (ATCC) were infected with 2x concentrated lentivirus by spinoculation in retronectin-coated (Takara) plates at 800g for 45 mins at 32°C. After infection for 2 days, the GFP-positive cells (NALM6-GL) were sorted on a BD FACSAria II. The second round sorting was performed after culture for two additional days. To test the luciferase expression in NALM6-GL, cells were incubated with 150μg/ml D-Luciferin (PerkinElmer) and bioluminescence signal intensity was measured by an IVIS system.

Dai X., Park J.J., Du Y., Kim H.R., Wang G., Errami Y, & Chen S. (2019). One-step generation of modular CAR-T with AAV-Cpf1. Nature methods, 16(3), 247-254.

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Enhancing Hematopoietic Progenitor Expansion

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Sorted CD34⁺CD45⁺ EB progenitors were seeded on RetroNectincoated (Takara; 10 μg/cm²) 96-well plates at a density of 2 × 10⁴ to 5 × 10⁴ cells per well in Serum-Free Expansion Medium (SFEM) (Stemcell) with SCF (50 ng/ml), FLT3 (50 ng/ml), thrombopoietin (TPO) (50 ng/ml), IL-6 (50 ng/ml), and IL-3 (10 ng/ml) (R&D). Lentiviral infections were carried out in a total volume of 150 μl. The multiplicity of infection (MOI) for ERG and HOXA9 is 5, whereas the MOI for RORA, SOX4, and MYB is 3. Virus was concentrated onto cells by centrifuging at 2500 rpm for 30 min. Infections were carried out for 24 hours. After gene transfer, CD34-5F cells were cultured in SFEM + SCF (50 ng/ml), FLT3 (50 ng/ml), TPO (50 ng/ml), IL-6 (50 ng/ml), and IL-3 (10 ng/ml) (R&D). Dox was added at 2 μg/ml (Sigma). Cultures were maintained at a density of <1 × 10⁶ cells/ml, and medium was changed every 3 to 4 days. After 14 days, CD34-5F cells were plated according to the erythroid protocol or transplanted in vivo.

Doulatov S., Vo L.T., Macari E.R., Wahlster L., Kinney M.A., Taylor A.M., Barragan J., Gupta M., McGrath K., Lee H.Y., Humphries J.M., DeVine A., Narla A., Alter B.P., Beggs A.H., Agarwal S., Ebert B.L., Gazda H.T., Lodish H.F., Sieff C.A., Schlaeger T.M., Zon L.I, & Daley G.Q. (2017). Drug discovery for Diamond-Blackfan anemia using reprogrammed hematopoietic progenitors. Science translational medicine, 9(376), eaah5645.

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Inducible Myeloid Leukemia Regression

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KSL cells were sorted (see Supplemental Experimental Procedures) from IDH2^R140Qhm-t mice that had been treated with doxycycline for 6 weeks. Cells were cultured for 8 hr in StemSpan serum free expansion medium (StemCell Technologies) supplemented with murine SCF, TPO, IL-3, IL-6, and Flt3-L (PeproTech) on Retronectin-coated (Takara Bio) petri dishes. Cells were transduced overnight using MSCV-HoxA9-GFP and MSCV-Meis1a–YFP (kindly provided by Dr. Christian Bach). The next day, cells were harvested and washed twice with phosphate buffered saline (PBS). Viable cells were counted following trypan blue staining and 5,000 cells were injected retro-orbitally into sublethally irradiated (6.5Gr) C57BL/6 recipients. For the initial experiment, secondary transplantation and randomized deinduction was performed as outlined in Figure 4A. For the repeat experiment, all recipients were maintained on doxycycline food for 2 weeks, at which time 2 recipients were analyzed to confirm presence of GFP/YFP⁺ cells in the bone marrow. Randomized deinduction was then performed with recipients randomly assigned to doxycycline-treated or untreated groups. Mice were then analyzed every 2 weeks to monitor the kinetics of disease regression.

Kats L.M., Reschke M., Taulli R., Pozdnyakova O., Burgess K., Bhargava P., Straley K., Karnik R., Meissner A., Small D., Su S.M., Yen K., Zhang J, & Pandolfi P.P. (2014). Proto-Oncogenic Role of Mutant IDH2 in Leukemia Initiation and Maintenance. Cell stem cell, 14(3), 329-341.

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Murine Anti-CD19 CAR T Cell Generation

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Retroviral supernatants were produced by transfection of 293GP cell line
using Lipofectamine 2000 (Life Technologies) with plasmids encoding the CD19 CAR
and pCL-Eco retroviral envelope DNA. Supernatants were collected 24, 48, and 72
hours after transfection. Construction of murine anti-CD19 CD28 CAR was
previously described (41 (link)). T cells were
extracted from murine splenocytes using a T cell enrichment column (R&D
Systems). Purified (>90%) CD4 or CD8 T cell subsets were separated using
untouched CD4 T Cell Isolation Kit or untouched CD8 T cell Isolation Kit
(Miltenyi Biotec) before activation. Cells were then activated using
anti-CD3/CD28 beads (Life Technologies) on day 1 using 3:1 beads/cell ratio with
purified T cell beads then removed on day 3 after transduction. Cells were
cultured with IL-2 (30 IU/ml) and IL-7 (10 ng/ml) for 5 days. Activated T cells
were transduced using RetroNectin-coated (Takara) plates using combined viral
supernatants on days 2 and 3. T cells were evaluated or infused on day 5.
Transduction efficiencies were routinely 60 to 90% for all CAR T cells used in
the experiments.

Yang Y., Kohler M.E., Chien C.D., Sauter C.T., Jacoby E., Yan C., Hu Y., Wanhainen K., Qin H, & Fry T.J. (2017). TCR engagement negatively affects CD8 but not CD4 CAR T cell expansion and leukemic clearance. Science translational medicine, 9(417), eaag1209.

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Autologous Hematopoietic Stem Cell Transplantation

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All animal studies were approved by the NHLBI Animal Care and Use Committee. Mobilization with G-CSF and stem cell factor (SCF), CD34⁺ cell collection and immunoselection, transduction, conditioning, and transplantation were performed as described (Donahue et al., 2005 ). CD34⁺ cells were cultured overnight on RetroNectin-coated (Takara, Otsu, Japan) plates in X-vivo 10 media (Lonza, Allendale, NJ) supplemented with 100 ng/ml each human Flt3 ligand, SCF, and thrombopoietin (R&D Systems, Minneapolis, MN), and 1% human albumin, and transduced with barcode-containing lentiviral vectors at a multiplicity of infection (MOI) of 25 in the presence of 4 μg/ml protamine sulfate (Sigma, St Louis, MO). 24 hours later, transduced CD34⁺ cells were reinfused into the irradiated (500cGy a day for two days) autologous macaque.

Wu C., Li B., Lu R., Koelle S.J., Yang Y., Jares A., Krouse A.E., Metzger M., Liang F., Loré K., Wu C.O., Donahue R.E., Chen I.S., Weissman I, & Dunbar C.E. (2014). Clonal Tracking of Rhesus Macaque Hematopoiesis Highlights A Distinct Lineage Origin for Natural Killer Cells. Cell stem cell, 14(4), 486-499.

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Retroviral Transformation of Murine Bone Marrow

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Bone marrow cells were extracted from young age-matched mice (Supplemental Table S3) and processed as described in the Supplemental Material. All mouse experiments were approved by the University of California at San Francisco Institutional Animal Care and Use Committee (IACUC). Bone marrow cells collected were retrovirally transformed with BCR-ABL1 in the presence of 10 ng/mL IL-7 (Peprotech) for Ph⁺ ALL-like cells or a cocktail of 10 ng/mL IL-3, 25 ng/mL IL-6, and 50 ng/mL SCF (PeproTech) for chronic myeloid leukemia-like cells on RetroNectin-coated (Takara) dishes. See the Supplemental Material for details.

Hurtz C., Chan L.N., Geng H., Ballabio E., Xiao G., Deb G., Khoury H., Chen C.W., Armstrong S.A., Chen J., Ernst P., Melnick A., Milne T, & Müschen M. (2019). Rationale for targeting BCL6 in MLL-rearranged acute lymphoblastic leukemia. Genes & Development, 33(17-18), 1265-1279.

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Activation and Transduction of CAR-T Cells

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Cryopreserved human peripheral blood mononuclear cells from three healthy donors were thawed and activated with anti-CD3 (clone OKT3) and anti-CD28 (clone 28.2) antibodies (Biolegend) and cultured with IL-7 (10 ng / mL) and IL-15 (5 ng / mL) (R&D System). PBMCS were transduced on day three with the RVSFG.CD19.CD28.4-1BBzeta retroviral vector (kindly provided by M Brenner, Baylor College of Medicine, Houston, Texas) using spinoculation on retronectin-coated (3.5 µg / mL, Takara) 24-well plates. CAR-T cells were maintained in cytokine-supplemented media (ImmunoCult™-XF T Cell Expansion Medium, Stemcell Technologies) and used for experiments 10 days after transduction.

Hunger J., Schregel K., Boztepe B., Agardy D.A., Turco V., Karimian-Jazi K., Weidenfeld I., Streibel Y., Fischer M., Sturm V., Santarella-Mellwig R., Kilian M., Jähne K., Sahm K., Wick W., Bunse L., Heiland S., Bunse T., Bendszus M., Platten M, & Breckwoldt M.O. (2023). In vivo nanoparticle-based T cell imaging can predict therapy response towards adoptive T cell therapy in experimental glioma. Theranostics, 13(15), 5170-5182.

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Retroviral and lentiviral transduction of T-cells

Cited 1 time

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Generation of ecotropic viral particles containing GFP, ITK-SYK or ITK-SYK^KD constructs and infection of Jurkat-Eco cells were performed as previously described8 (link). To generate amphotropic retrovirus, Phoenix-Ampho cells were grown in 15 cm dishes and transfected using FuGENE (Promega, Madison, Wisconsin, USA) with 10 μg ITK-SYK or ITK-SYK^KD8 (link). The media was changed 8 hours after transfection. The viral supernatant was collected 36 hours after transfection, passed through a 0.45 μm filter and used fresh or snap-frozen for later. Activated primary human CD4 T-cells were spinfected with RetroNectin-coated (Takara Bio Inc.) 6-well plates (5 mg/ml). For the production of lentiviral particles, we used HEK293FT cells. FuGENE-based transfection was performed with 17 μg pCMV delta R8.2, 10 μg pHCMV-10A1 and 10 μg PD-1 or PD-1 YFYF. HH cells were spinfected with the lentivirus on RetroNectin-coated 6-well plates (5 mg/ml).

Wartewig T., Kurgyis Z., Keppler S., Pechloff K., Hameister E., Öllinger R., Maresch R., Buch T., Steiger K., Winter C., Rad R, & Ruland J. (2017). PD-1 is a haploinsufficient suppressor of T cell lymphomagenesis. Nature, 552(7683), 121-125.

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Generation of BK Cells for CRISPR Screening

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Sorted KL cells or single-cell suspensions from the spleens of tumour bearing Bcor^ΔE9-10Kras^G12D mice (referred to as BK cells) were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with penicillin/streptomycin, 20% FCS, L-Asparagine (0.1mM), IL-3 (2ng/ml), IL-6 (2ng/ml), and SCF (10ng/ml). Cells were maintained at 37 °C and 10% CO₂. Cells were genotyped as above to confirm their identity and were tested for Mycoplasma contamination. For transduction of BK cells, lentiviral supernatants were harvested from packaging 293T cells transfected with the custom CRISPR library (see below). Lentiviral particles were centrifuged onto RetroNectin-coated (Takara) non-treated tissue-culture plates and virus infection performed according the manufacturer’s protocol.

Kelly M.J., So J., Rogers A.J., Gregory G., Li J., Zethoven M., Gearhart M.D., Bardwell V.J., Johnstone R.W., Vervoort S.J, & Kats L.M. (2019). Bcor loss perturbs myeloid differentiation and promotes leukaemogenesis. Nature Communications, 10, 1347.

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Retroviral Transduction of Mouse LSK Cells

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MSCV-Ras-pgk-puro was created by subcloning human NRASQ61K sequences (Addgene plasmid 12543, Channing Der lab) as a BamH1 fragment into the MSCV-puro vector. See Supplemental Experimental Procedures for published plasmids. LIN⁻SCA1 ⁺KIT⁺ (LSK-) cells were sorted from donor bone marrow using a MoFlo flow sorter (Beckman Coulter) were prestimulated in liquid culture in the presence of murine SCF (20 ng/ml), FLT3L (20 ng/ml), IL6 (20 ng/ml), and TPO (10 ng/ml) (Peprotec) and then transduced on Retronectin-coated (Takara) plates. Cells were sorted for expression of GFP or YFP and were selected in the presence of Puromycin on OP9-DL1 cells carrying a resistance gene for puromycin in the presence of FLT3L (5 ng/ml), SCF (5 ng/ml), and IL7 (1 ng/ml). Cells expanded for 14–21 days were then injected into syngeneic sublethally (600 cGy) irradiated recipients. Cell growth and viability were followed by serial cell counts. Antibodies used for flow cytometry and immunoblot detection and qPCR primers are detailed in Supplemental Experimental Procedures.

Danis E., Yamauchi T., Echanique K., Zhang X., Haladyna J.N., Riedel S.S., Zhu N., Xie H., Orkin S.H., Armstrong S.A., Bernt K.M, & Neff T. (2016). Ezh2 Controls an Early Hematopoietic Program and Growth and Survival Signaling in Early T Cell Precursor Acute Lymphoblastic Leukemia. Cell reports, 14(8), 1953-1965.

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