Quicktiter lentivirus quantitation kit

Manufactured by Cell Biolabs

Sourced in United States

The QuickTiter Lentivirus Quantitation Kit is a laboratory tool designed to quantify the titer of lentiviral particles. It provides a rapid and reliable method for determining the concentration of lentivirus in a sample.

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Lab products found in correlation

Peg it virus precipitation solution, by System Biosciences (2 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions) Pmd18 t vector, by Takara Bio (1 mentions) Lenti xtm concentrator, by Takara Bio (1 mentions) Plenticmv gfp neo, by Addgene (1 mentions) Pspax2, by Addgene (1 mentions) Polybrene, by Merck Group (1 mentions) Puromycin, by Merck Group (1 mentions) 24 well plate, by Corning (1 mentions) Pei reagent, by Merck Group (1 mentions) Lentiviral vectors plko 1, by Thermo Fisher Scientific (1 mentions) Polyfect, by Qiagen (1 mentions) 0.45 μm filter, by Merck Group (1 mentions)

Close spelling variants of this product

QuickTiter HIV Lentivirus Quantitation Kit

9 protocols using quicktiter lentivirus quantitation kit

Lentiviral transduction of NK-92MI cells

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The plasmid pGMLV-CMV-CD16-EF1-GFP-T2A-Puro was purchased from Shanghai Genomeditech Company, and then the sequence was verified by Sanger sequencing. GFP was inserted as a fluorescent marker to determine the infection efficiency and for gating in the flow cytometric analysis. The puromycin-resistant gene was the selective marker of this vector for the selection and enrichment of GFP-positive clones. The lentivirus was generated by cotransfecting 80% confluent HEK293T cells with psPAX2 and pMD2G using Lipofectamine 3000 (Invitrogen). The QuickTiter Lentivirus Quantitation Kit (Cell Biolabs, Inc.) was used to detect the virus titer according to a previous reference.54 (link)
A total of 3×10⁵ NK-92MI cells were seeded into a 48-well plate in a final volume of 100 μL, and then lentivirus was added at a multiplicity of infection of 500. At the same time, polybrene was also added to a final concentration of 10 µg/mL. Twenty-four hours later, the virus solution was replaced by the fresh complete culture medium. After an additional 24 hours, puromycin at a final concentration of 2 μg/mL was supplemented for continuous selection. After 7 days of selection, the cells were subject to flow cytometry (FC) to determine the infection efficiency.

Wang H., Yang J., Pan H., Tai M.C., Maher M.H., Jia R., Ge S, & Lu L. (2020). Dinutuximab Synergistically Enhances the Cytotoxicity of Natural Killer Cells to Retinoblastoma Through the Perforin-Granzyme B Pathway. OncoTargets and therapy, 13, 3903-3920.

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Cloning and Lentiviral Production of Mouse and Human Chemerin

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A mouse chemerin cDNA made from groin fat RNA was used to amplify the chemerin coding sequence using the following primer set: forward primer 5′-ACCGAATTCAGGTGAAGCCATGAAGTGCT-3′, with an EcoRI restriction site, and reverse primer 5′-TGCGGCCGCCTGTCTAGGGCTTATTTG-3′, with a NotI restriction site. The chemerin fragment was then cloned into the pCDH-CMV-MCS-EF1-copGFP (CD511B-1) vector (LV-GFP, System Biosciences, U.S.A.) to produce LV-mChemerin-GFP. Similarly, the human chemerin coding sequence was amplified from JEG-3 cells cDNA by using the following primer set: forward primer, 5′-TGGAAGAAACCCGAGTGCAAA-3′ and reverse primer, 5′- AGAACTTGGGTCTCTATGGGG-3′. The PCR product was ligated into the pMD18-T Vector (Takara Bio Inc., Shiga, Japan), after which the chemerin coding sequence was liberated by digestion with EcoRI and NotI and cloned into LV-GFP to produce LV-hChemerin-GFP. The constructs were transfected into packaging HEK293T cells as previously described to produce lentiviral particles [17 (link),18 (link)], and the lentivirus-containing supernatant was harvested and tittered using the QuickTiter Lentivirus quantitation kit (Cell Biolabs Inc, San Diego, CA, U.S.A.).

Tan L., Chen Z., Sun F., Zhou Z., Zhang B., Wang B., Chen J., Li M., Xiao T., Neuman R.I., Niu J., Verdonk K., Lu X., Zhang J.V., Danser A.H., Yang Q, & Fan X. (2022). Placental trophoblast-specific overexpression of chemerin induces preeclampsia-like symptoms. Clinical Science (London, England : 1979), 136(4), 257-272.

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Lentiviral Vector Construction and Quantification

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The cDNAs of human HCN4, human wild type (SGO1-WT, NM_001199252.3) and mutant (SGO1-K23E) SGO1 were subcloned into the lentiviral vector pLentiCMV in which the GFP coding section was deleted from the backbone of pLentiCMV-GFP-Neo (Addgene #17447, gift from Eric Campeau & Paul Kaufman). The GFP was cloned into the N-terminal of HCN4 and mCherry was cloned into the N-terminal of SGO1-WT and SGO1-K23E to form fusion proteins. The lentiviral plasmids carrying SGO1-WT, SGO1-K23E, HCN4, SGO1-WT-mCherry, SGO-K23E-mCherry, or HCN4-GFP were co-transfected into HEK293T cells with packaging plasmids pMD2.G (Addgene #12259) and psPAX2 (Addgene #12260), which were gifts from Didier Trono. The supernatants containing virus were harvested 48 and 72 h after transfection and concentrated using Lenti-X^TM concentrator (TaKaRa Bio). The viral pellet was resuspended in serum-free DMEM medium and viral titers were measured with QuickTiter Lentivirus Quantitation Kit (Cell Biolabs). Viral stocks were stored at –80 °C until use.

Liu D., Song A.T., Qi X., van Vliet P.P., Xiao J., Xiong F., Andelfinger G, & Nattel S. (2021). Cohesin-protein Shugoshin-1 controls cardiac automaticity via HCN4 pacemaker channel. Nature Communications, 12, 2551.

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Lentiviral Knockdown of LRP1 in Megakaryocytes

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Three shRNAs against mouse LRP1 were created in a lentivirus that conferred puromycin resistance (EZBiolab). Plasmid DNA for vectors was produced in DH5α bacteria, and using a 3-vector lentivirus system, viral particles were generated in 293T cells as described(30 (link)). Virus was titered by the QuickTiter Lentivirus Quantitation Kit (Cell Biolabs), yielding titers between 0.9–1.8 × 10¹¹ viral particles/ml. Using primary BM obtained from Pf4^−/− mice, cells were pelleted and resuspended at a concentration of 2 × 10⁶ cells/ml in unmodified IMDM. Approximately 500 μl of the cell suspension was placed in a 24-well plate (Corning), and plates were spun at 340g for 5 minutes to form a monolayer of cells. Then, 4 μl of polybrene (2 mg/ml, Millipore) was added to cells with 50 μl of concentrated virus. Cells were spun again at 340g for 90 minutes at 20°C and then placed at 37°C for 3–4 hours. Afterwards, cells were resuspended in fresh serum-free AIM-V media with 50-ng/ml recombinant TPO. One μg/ml puromycin (Sigma) was added after 48 hours. After 3 days, hPF4 (25 μg/ml) was added to the culture media for 24 hours, and cell lysates were prepared and analyzed as described under culture of megakaryocytes.

Lambert M.P., Meng R., Xiao L., Harper D.C., Marks M.S., Kowalska A.M, & Poncz M. (2015). Intramedullary megakaryocytes internalize released platelet factor 4 (PF4) and store it in alpha granules. Journal of thrombosis and haemostasis : JTH, 13(10), 1888-1899.

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Lentivirus Production and Quantification

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All of the lentiviruses were generated in HEK293T[mEF-2(G717R)] after transient transfection with the PEI reagent (Sigma, Saint Louis, USA, Cat No. 40872-7) and plasmids (Figure S3 http://links.lww.com/MD/A359). Lentivirus-containing medium was collected every 24 h for 3 days, and cellular debris was cleared by low-speed centrifugation and passage through a 0.45-μm filter (Millipore, Billerica, USA, Cat No. SLHV033RB). The collected medium was concentrated with the PEG-it™ virus precipitation solution (SBI, Mountain View, USA, Cat No. LV810A-1), and pellets containing viral particles were re-suspended with DMEM. Lentiviral titers was measured with the QuickTiter™ Lentivirus Quantitation Kit (CELL BIOLABS, San Diego, USA, Cat No. VPK-108-HIV-P24).

Lin B., Gao A., Zhang R., Ma H., Shen H., Hu Q., Zhang H., Zhao M., Lan X, & Liu K. (2015). Use of a Novel Integrase-Deficient Lentivirus for Targeted Anti-Cancer Therapy With Survivin Promoter-Driven Diphtheria Toxin A. Medicine, 94(31), e1301.

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Lentiviral Transduction of EZH2 Knockdown

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Lentiviral vectors (pLKO.1) were purchased from Openbiosystems and calcium phosphate transfection of HEK293T packaging cells was used to make virus as described previously [50 (link)]. A non-targeting (shNT; #RHS6848) vector was used as a control. The target sequence of the vectors is as follows: shEZH2_074 (5′-GCTAGGTTAATTGGGACCAAA-3′), shEZH2_ 474 (5′-CAACACAAGTCATCCCATTAA-3′). Concentrated viral titers were determined using the QuickTiter Lentivirus Quantitation Kit (Cell Biolabs Inc, San Diego, CA, USA). Cells were transfected with virus in the presence of Polybrene (8 μg/mL) and selected with puromycin (1 μg/mL) 48 hr after transfection for an additional 96 hr.

Tiffen J.C., Gunatilake D., Gallagher S.J., Gowrishankar K., Heinemann A., Cullinane C., Dutton-Regester K., Pupo G.M., Strbenac D., Yang J.Y., Madore J., Mann G.J., Hayward N.K., McArthur G.A., Filipp F.V, & Hersey P. (2015). Targeting activating mutations of EZH2 leads to potent cell growth inhibition in human melanoma by derepression of tumor suppressor genes. Oncotarget, 6(29), 27023-27036.

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Lentiviral Transduction of Immune Cells

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DAP12 ITAM mutant forms Y91F, Y102F and Y91F/102F (2YF) were generated by site
directed mutagenesis (Stratagene) and clones were confirmed by sequencing. The majority of
hematopoietic cells are non-dividing or slowly self-renewing; thus refractory to most
non-viral or retroviral delivery methods. Since lentiviral vectors are capable of
transducing non-dividing cells and maintaining long-term and sustained expression of the
transgenes we used HIV-1 derived vector system with minor modifications(29 (link)). Human wild-type DAP12 and DAP12 mutants containing the
Xpress peptide epitope (DLYDDDDK), were subcloned into the pCCLsin.PPT.hEF1a.pre, vector
(Sanjeev Gupta, Albert Einstein College of Medicine, NY, USA). To generate the retrovirus
HEK293T cells were cotransfected with pCCL constructs and packaging plasmids pMDLg/pRRE,
pRSV-Rev and pMD2.VSV-G using polyfect, (QIAGEN). Viral titers of 5×10⁶viral particles measured by QuickTiter^™ Lentivirus Quantitation Kit
(Cell biolabs, USA) were used for optimal infection. This viral titer had no cytotoxic
effects on target cells. The virus-containing supernatant was collected 72 hrs after
transfection and added to PBMC plated on 10 cm culture dishes. After 48 hrs, the viral
supernatant was replaced with fresh medium. Cells were stimulated with IL-23 as described
above.

SHIN H.S., SARIN R., DIXIT N., WU J., GERSHWIN M.E., BOWMAN E.P, & ADAMOPOULOS I.E. (2014). Crosstalk among interleukin 23 and DNAX activating protein 12-dependent pathways promotes osteoclastogenesis. Journal of immunology (Baltimore, Md. : 1950), 194(1), 316-324.

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Lentiviral Vector Encoding Rat MK

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A lentiviral vector system was selected because lentiviruses exhibit limited toxicity to infected cells. To construct the lentivirus encoding MK plasmid (pLenO-DCE-MK), the cDNA-encoding rat MK (NM_030859, 423-bp cDNA) was synthesized and cloned into the EcoRI and BamHI restriction endonuclease sites of the pLenO-DCE vector (cat. No. 26208-1, Invabio, Shanghai, China), a mammalian expression vector containing green fluorescent protein (GFP) and puromycin resistance genes. After the correct sequence was confirmed, lentiviral vector particles were produced in accordance with the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). pRsv-REV, pMDlg-pRRE, pMD2G and pLenO-DCE-MK (or pLenO-DCE) were co-transfected into 293 T cells, and viral supernatants were harvested 48 and 72 hours after transfection, passed through 0.45-μm filters (Millipore corp., Bedford, Massachusetts, USA) and concentrated using four rounds of ultracentrifugation [23 (link)]. The viral pellet was resuspended in serum-free Dulbecco’s modified Eagle’s medium to obtain a 10,000-fold concentrate, and the debris was spun down. Viral stocks were stored at -80°C until use for transduction; functional viral titers were measured using QuickTiter Lentivirus Quantitation Kit (Cell Biolabs, San Diego, California, USA) and were determined by infection of 293 T cells [24 (link)].

Zhao S.L., Zhang Y.J., Li M.H., Zhang X.L, & Chen S.L. (2014). Mesenchymal stem cells with overexpression of midkine enhance cell survival and attenuate cardiac dysfunction in a rat model of myocardial infarction. Stem Cell Research & Therapy, 5(2), 37.

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Lentiviral Induction of Myogenic Differentiation

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GRMD MABs were transduced with a lentiviral vector containing a tamoxifen-inducible MyoD-ER expression cassette (61 (link)) to induce myogenic differentiation at late passages (i.e. passage P15–18). Lentiviral vector titration was calculated based upon the p24 assay (QuickTiter Lentivirus Quantitation Kit, Cellbiolabs). Working concentrations were determined by assessing myogenic differentiation efficiency at increasing multiplicities of infection (MOI). Briefly, 0.14 × 10⁶ cells were transduced with 1, 5 or 50 MOI of MyoD-ER lentiviral vector in 1 ml of culture medium and incubated for 12 h at 37°C in a 5% CO₂, 3% O₂ cell culture incubator. Media were subsequently changed and cells maintained in culture for 2–3 passages, after which the in vitro differentiation assay into skeletal muscle was performed (see specific section below). The optimal condition was set at 50 MOI.

Loperfido M., Jarmin S., Dastidar S., Di Matteo M., Perini I., Moore M., Nair N., Samara-Kuko E., Athanasopoulos T., Tedesco F.S., Dickson G., Sampaolesi M., VandenDriessche T, & Chuah M.K. (2015). piggyBac transposons expressing full-length human dystrophin enable genetic correction of dystrophic mesoangioblasts. Nucleic Acids Research, 44(2), 744-760.

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