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Retro x qrt pcr titration kit

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The Retro-X™ qRT-PCR Titration Kit is a laboratory tool designed to quantify retroviral titer in cell culture supernatants using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) technology.

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Lab products found in correlation

Puromycin, by R&D Systems (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Puromycin, by Thermo Fisher Scientific (1 mentions) Geneart, by Thermo Fisher Scientific (1 mentions) Recombinant murine il 3, by R&D Systems (1 mentions) Penicillin streptomycin glutamine (psg), by Thermo Fisher Scientific (1 mentions) Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions) Lipofectamine ltx kit, by Thermo Fisher Scientific (1 mentions)

5 protocols using retro x qrt pcr titration kit

1

Retroviral CAR Transduction of NK and T Cells

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The retroviral plasmid was designed to carry the cassette of a second-generation CAR with specificity for CD123. A single-chain variable fragment (scFv), derived from a murine 7G3 antibody, was linked via an optimized human CD8 hinge-transmembrane domain to the costimulatory domains 4-1BB (CD137) and CD3-ζ. As a trackable marker, we added a peptide derived from the human phosphoglycoprotein CD34 (ΔCD34) [27 ].
Retroviral supernatant was generated in 293 T cells [28 (link)] and the viral particles (VPs) was quantified using a Retro-X™ qRT-PCR Titration Kit (Takara). NK and T cells were transduced using 109 VPs/0.5 × 106 cells.
Caruso S., De Angelis B., Del Bufalo F., Ciccone R., Donsante S., Volpe G., Manni S., Guercio M., Pezzella M., Iaffaldano L., Silvestris D.A., Sinibaldi M., Di Cecca S., Pitisci A., Velardi E., Merli P., Algeri M., Lodi M., Paganelli V., Serafini M., Riminucci M., Locatelli F, & Quintarelli C. (2022). Safe and effective off-the-shelf immunotherapy based on CAR.CD123-NK cells for the treatment of acute myeloid leukaemia. Journal of Hematology & Oncology, 15, 163.
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2

CAR.CD30 T Cell Transduction Protocol

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The scFv for CAR.CD30 molecules is derived from the AC10 monoclonal antibody.25 (link) The details of the constructs are provided in Figure 1A, Online Supplementary Figure S1A, Online Supplementary Materials and Online Supplementary Table S1, which report the amino-acid sequences for all the construct components. Retroviral supernatant was generated in 293T-cells19 (link),26 (link),27 (link) and quantified using a Retro-X™ qRT-PCR Titration Kit (Takara) to be used at 109 retrovirus-copies/0.5x106 T cells. The supernatant was used to transduce primary T cells derived from peripheral blood mononuclear cells of healthy donors (Ethical Committee approval n. 969/2015 protocol n. 669LB). More details on transduction are provided in the Online Supplementary Materials.
Guercio M., Orlando D., Di Cecca S., Sinibaldi M., Boffa I., Caruso S., Abbaszadeh Z., Camera A., Cembrola B., Bovetti K., Manni S., Caruana I., Ciccone R., Del Bufalo F., Merli P., Vinti L., Girardi K., Ruggeri A., De Stefanis C., Pezzullo M., Giorda E., Scarsella M., De Vito R., Barresi S., Ciolfi A., Tartaglia M., Moretta L., Locatelli F., Quintarelli C, & De Angelis B. (2020). CD28.OX40 co-stimulatory combination is associated with long in vivo persistence and high activity of CAR.CD30 T cells. Haematologica, 106(4), 987-999.
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3

Genetic Modification of T, NK, and B Cells

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T and NK cells from HD, as well as the B-cell leukemia/lymphoma cell lines DAUDI and NALM-6, were genetically modified using the retroviral construct iC9.CAR.CD19 (1e9 retrovirus-copies/0.5×106 cells. Multiplicity of infection were quantified by Retro-X™ qRT-PCR Titration Kit from Takara, USA). The bicistronic CAR construct carries antihuman CD19-scFv from FMC63, CD8 stalk domain, CD8 transmembrane domain, 4.1bb, and CD3ζ cytoplasmic domains cloned in-frame with the iC9 suicide gene (3 (link)).
Guercio M., Manni S., Boffa I., Caruso S., Di Cecca S., Sinibaldi M., Abbaszadeh Z., Camera A., Ciccone R., Polito V.A., Ferrandino F., Reddel S., Catanoso M.L., Bocceri E., Del Bufalo F., Algeri M., De Angelis B., Quintarelli C, & Locatelli F. (2021). Inclusion of the Inducible Caspase 9 Suicide Gene in CAR Construct Increases Safety of CAR.CD19 T Cell Therapy in B-Cell Malignancies. Frontiers in Immunology, 12, 755639.
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4

Retroviral Transduction of BaF3 Cells with FGFR3

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Murine pro-B lymphocyte BaF3 cells (DSMZ, Germany) were transduced with retroviral particles to stably overexpress either human FGFR3 IIIc or FGFR3 IIIc G380R. Briefly, GP-2 HEK packaging cell line (Clontech) was co-transfected with a pantropic VSVg vector (Clontech) and a pBABE-puro-hFGFR3 IIIc or hFGFR3 IIIc G380R (GeneArt, Thermo Fisher Scientific) using a standard CaCl2 procedure. After 48h, supernatant containing retroviral particles were harvested and viral RNA copy was determined by RT-qPCR (Retro-X™ qRT-PCR Titration Kit, Clontech).
BaF3 were then transduced by spinocculation (centrifugation 800g, 30 minutes at 32°C) and puromycin (Gibco) was added 3 days later to start the selection. 2μg/mL final puromycin concentration was chosen based on a puromycin kill curve made with non-transfected BaF3.
BaF3 mutant cells were cultured in RPMI-1640 medium (Gibco) supplemented with 10% heat inactivated fetal bovine serum (Gibco), 1% Penicillin/Streptomycin/Glutamine (Gibco), 10ng/mL recombinant murine IL-3 (R&D Systems) and 2μg/mL puromycin.
Gonçalves D., Rignol G., Dellugat P., Hartmann G., Sarrazy Garcia S., Stavenhagen J., Santarelli L., Gouze E, & Czech C. (2020). In vitro and in vivo characterization of Recifercept, a soluble fibroblast growth factor receptor 3, as treatment for achondroplasia. PLoS ONE, 15(12), e0244368.
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5

Retroviral Transduction of PEDF

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The human PEDF cDNA (NotI/ClaI) was cloned into the retroviral bicistronic pRetroX-IRES-DsRed Express plasmid (Clontech). PT67 cells were co-transfected with 1 μg pPUR (Clontech) and 10 μg of either pRetroX-IRES-DsRed Express or pRetroX-PEDF-IRES-DsRed Express using the Lipofectamine LTX kit (Invitrogen, Carlsbad, CA, USA). Transfectant single clones were selected in 3 μg/ml puromycin. Viral titration was determined using the Retro-X qRT-PCR titration kit (Clontech).
Nelius T., Martinez-Marin D., Hirsch J., Miller B., Rinard K., Lopez J., de Riese W, & Filleur S. (2014). Pigment epithelium-derived factor expression prolongs survival and enhances the cytotoxicity of low-dose chemotherapy in castration-refractory prostate cancer. Cell Death & Disease, 5(5), e1210-.
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