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Lentivirus qpcr titer kit

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The Lentivirus qPCR Titer Kit is a laboratory tool designed for quantifying the titer of lentiviral particles. It utilizes a quantitative real-time PCR (qPCR) method to determine the viral genome copy number, providing a precise and reliable measurement of lentivirus concentration.

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Lab products found in correlation

Peg it, by System Biosciences (18 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (6 mentions) Pspax2, by Addgene (5 mentions) Opti mem, by Thermo Fisher Scientific (5 mentions) Pmd2 g, by Addgene (4 mentions) Pcdh cmv mcs ef1a rfp, by System Biosciences (2 mentions) Lipofectamine 2000, by Roche (2 mentions) Pei max, by Polysciences (2 mentions) Pcmv dr8, by Addgene (1 mentions) Lenti x concentrator kit, by Takara Bio (1 mentions) Pcmv vsv g envelope, by Addgene (1 mentions) Endotoxin free maxiprep, by Qiagen (1 mentions) Luciferase assay reagent, by Promega (1 mentions) Reporter lysis buffer, by Promega (1 mentions) Fluostar omega microplate reader, by BMG Labtech (1 mentions) Transit lt1 transfection reagent, by Mirus Bio (1 mentions) Tet plko puro, by Addgene (1 mentions) Misson shrna, by Merck Group (1 mentions) Mission shrna constructs, by Merck Group (1 mentions) Lenti x maxi purification kit, by Takara Bio (1 mentions)

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QPCR Lentivirus Titration (Titer) Kit Lentivirus Titer Kit Lentivirus

27 protocols using lentivirus qpcr titer kit

1

Lentiviral Transduction of Ceg3 in COS-1 Cells

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To produce lentivirus for transduction expression of Ceg3 in COS-1 cells, Ceg3 was inserted into pCDH-CMV-MCS-EF1a-RFP (System Biosciences cat# CD512B-1), which was transfected together with pMD2.G (Addgene plasmid #12259) and psPAX2 (Addgene plasmid #12260) vector (Salmon and Trono, 2007 (link)) into HEK293T cells grown to about 70% confluence. Supernatant was collected after 48 hr and then filtered with a 0.45-μm syringe filter. The titer of the produced lentivirus was determined by using qPCR Lentivirus Titer Kit (abm, cat# LV900). For lentiviral transduction, approximately 1×105 COS-1 cells seeded into 24-well plates 1 day before transduction were transduced with lentiviral particles at an MOI of 10. Cells incubated for 2 days at 37°C with 5% CO2 were collected for immunoblotting.
Fu J., Zhou M., Gritsenko M.A., Nakayasu E.S., Song L, & Luo Z.Q. (2022). Legionella pneumophila modulates host energy metabolism by ADP-ribosylation of ADP/ATP translocases. eLife, 11, e73611.
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2

Lentivirus Production and Delivery for Obesity Study

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Recombinant lentiviruses were produced by co-transfecting HEK 293T cells with the transfer vector (CCL) and helper plasmids (psPAX2 and pMD2. G) using Lipofectamine 2000 transfection reagent. A total of 5 × 106 293T cells were seeded in 10-cm plates for 24 h before transfection in DMEM with 10% fetal bovine serum (FBS), penicillin (100 IU/mL), and streptomycin (100 mg/mL) in a 5% CO2 incubator. The culture medium was changed one hour before transfection. A total of 64 μg of plasmid DNA, including 16 μg of the envelope plasmid Ppax2, 16 μg of the packing plasmid pMD2.G, and 32 μg of the transfer vector plasmid, were used per dish for transfection. After the lentiviruses were collected, we used the Lentivirus Purification Kit (ABM, G171) to purify the lentiviruses and increase their titers. The viral titer was determined by the qPCR Lentivirus Titer Kit (ABM, LV900). Mice aged 8–12 weeks were used in the experiments. To induce obesity, WT mice were fed an HFD for 18 weeks. For eWAT injections, control, shCCL5/shCCR5 or CCL5/CCR5 lentivirus (1 × 1010 ifu/mouse) was injected directly into bilateral eWAT in a total volume of 50 μL.
Chan P.C., Lu C.H., Chien H.C., Tian Y.F, & Hsieh P.S. (2022). Adipose Tissue-Derived CCL5 Enhances Local Pro-Inflammatory Monocytic MDSCs Accumulation and Inflammation via CCR5 Receptor in High-Fat Diet-Fed Mice. International Journal of Molecular Sciences, 23(22), 14226.
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3

Retroviral and Lentiviral Transduction Protocols

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Replication-defective high-titer retrovirus expressing v-RasHa was generated from ψ2-producer cells as described previously26 (link). Primary or immortalized mouse keratinocytes were transduced with the retrovirus in keratinocytes media containing 4 μg/ml polybrene after 3 days of culture. For lentivirus production, HEK293T cells were grown to ∼60% confluence in DMEM complete media supplemented with 1 mM solidum pyruvate, 8.93 mM sodium bicarbonate, 1X NEAA, 1X GlutaMAX, and 10 mM HEPES. pCW57.1 lentiviral vector containing human wildtype IRE1α sequence was co-transfected with pMD2.G envelope (Addgene #12259) and psPAX2 packaging (Addgene #12260) vectors in a molar ratio of 2:1:1 for 6h before replacing the transfection media. pWPI-Xbp1s26 (link) and pLox-Ttag-iresTK (Addgene #12246) vectors were co-transfected with the pMD2.G and psPAX2 vectors in a molar ratio of 1:1:1. Lentiviral particles in the supernatant were harvested 48 and 96h post-transfection and incubated overnight with sterile 10% w/v PEG 6000 in 2.5 M NaCl at 4°C. Lentivirus was concentrated by centrifugation at 10,000 rpm for 2h at 4°C. Pellet was resuspended in sterile ice-cold PBS. Lentivirus titer was estimated by qPCR lentivirus titer kit (abm) according to manufacturer’s instructions.
Mogre S., Blazanin N., Walsh H., Ibinson J., Minnich C., Hu C.C, & Glick A.B. (2022). TGFβ1 regulates HRas-mediated activation of IRE1α through the PERK-RPAP2 axis in keratinocytes. Molecular carcinogenesis, 61(10), 958-971.
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4

Lentiviral Transduction and Titer Quantification

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Lentivirus aliquots were thawed on ice and 3 μl of virus was used to transduce wells of 1.5 × 10^5 293T cells that were allowed to multiply to approximately 10^6 cells before the DNA were harvested 48 h later. A qPCR Lentivirus Titer Kit (ABM, LV900) was used to compare amplification of viral DNA to amplification of an endogenous gene (hVAR). See (Lerchner et al., 2023 ) for a detailed description of the procedure. For surgeries and as a starting point for the time course experiment, virus titers were adjusted to 2 × 10^9 iu/ml with cold PBS after thawing virus aliquots.
Lerchner W., Dash K., Rose D., Eldridge M.A., Rothenhoefer K.M., Yan X., Costa V.D., Averbeck B, & Richmond B.J. (2023). Efficient viral expression of a chemogenetic receptor in the old-world monkey amygdala. Current Research in Neurobiology, 4, 100091.
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5

Lentivirus Production for Ceg3 Expression in COS-1 Cells

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To produce lentivirus for transduction expression of Ceg3 in COS-1 cells, Ceg3
was inserted into pCDH-CMV-MCS-EF1a-RFP (System Biosciences cat# CD512B-1), which was transfected together with pMD2.G (Addgene plasmid #12259) and psPAX2
(Addgene plasmid #12260) vector ( 76) into HEK293T cells grown to about 70% confluence. Supernatant was collected after 48 h and then filtered with a 0.45 μm syringe filter. The titer of the produced lentivirus was determined by using qPCR Lentivirus Titer Kit (abm, cat# LV900). For lentiviral transduction, approximately 1x10 5 COS-1 cells seeded into 24-well plates 1 day before transduction were transduced with lentiviral particles at an MOI of 10. Cells incubated for 2 days at 37°C with 5% CO 2 were collected for immunoblotting.
Fu J., Zhou M., Gritsenko M., Nakayasu E., Song L., & Luo Z. (2021). Legionella pneumophila modulates host energy metabolism by ADP-ribosylation of ADP/ATP translocases.
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6

Lentiviral Vector Production and Transduction

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Expression vector plasmids were co-transfected with psPAX2 (Addgene plasmid 12260) and envelope vector pMD2.G (Addgene plasmid 12259) into 293T cells. Lentivirus was collected from the supernatant 48 and 72 hours after transfection. Stable cell lines were made by transducing cells with lentivirus at multiplicities of infection (MOIs) of 1, 0.3 (mini-library), and 0.1 (full library). Positive integrants were selected by treating with puromycin (hPGK-PuroR-P2A-HSV-TK) or flow sorting for mCherry (hPGK-HSV-TK-P2A-mCherry). Corresponding non-transduced cells for each cell line were used as positive control for puromycin selection. Lentiviruses for direct intratumoral delivery of HSV-TK interrupted by synMTERFD3i1-150-v6700 were 200X concentrated by ultracentrifugation (19,000g, 100 min at 4°C), resuspended in Opti-MEM (Gibco) and quantified using a qPCR Lentivirus Titer Kit (Applied Biological Materials).
North K., Benbarche S., Liu B., Pangallo J., Chen S., Stahl M., Bewersdorf J.P., Stanley R.F., Erickson C., Cho H., Pineda J.M., Thomas J.D., Polaski J.T., Belleville A.E., Gabel A.M., Udy D.B., Humbert O., Kiem H.P., Abdel-Wahab O, & Bradley R.K. (2022). Synthetic introns enable splicing factor mutation-dependent targeting of cancer cells. Nature biotechnology, 40(7), 1103-1113.
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7

Lentiviral Vector Production Protocol

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Lentiviruses were produced by co-transfecting HEK293T cells on 10-cm dishes with pLKO.1puro transfer, the pCMV-VSV-G envelope and pCMV-dR8.2 packaging plasmids (gift from Dr. Bob Weinberg, [25 (link)], purchased from Addgene) using the calcium phosphate precipitation method. In summary, plasmid DNAs were mixed in sterile distilled water, then 2.5 M CaCl2 was added (final concentration: 125 mM) and the solution was mixed dropwise with 2× HEPES-buffered solution [HBS] (42 mM HEPES, 15 mM D-glucose, 1.4 mM Na2HPO4, 10 mM KCl, 274 mM NaCl 274 mM, pH 7.1). This mixture was added dropwise to attached cells and the medium was replaced with fresh complete DMEM after 6 h. After 48 h had passed post-transfection, the cell medium was collected and centrifuged for 10 min at 3000 rpm, the supernatant was filtered and the lentiviral vector particles were purified and concentrated with a Lenti-X concentrator kit (Takara). After concentration, the viral particles were resuspended in sterile phosphate-buffered saline and the titer of the samples was measured with a qPCR Lentivirus titer kit from Applied Biological Materials (Vancouver, Canada). The samples were stored at −80 °C until the infection of cells.
Gém J.B., Kovács K.B., Szalai L., Szakadáti G., Porkoláb E., Szalai B., Turu G., Tóth A.D., Szekeres M., Hunyady L, & Balla A. (2021). Characterization of Type 1 Angiotensin II Receptor Activation Induced Dual-Specificity MAPK Phosphatase Gene Expression Changes in Rat Vascular Smooth Muscle Cells. Cells, 10(12), 3538.
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8

Lentiviral Vector Production and Transduction

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Expression vector plasmids were co-transfected with psPAX2 (Addgene plasmid 12260) and envelope vector pMD2.G (Addgene plasmid 12259) into 293T cells. Lentivirus was collected from the supernatant 48 and 72 hours after transfection. Stable cell lines were made by transducing cells with lentivirus at multiplicities of infection (MOIs) of 1, 0.3 (mini-library), and 0.1 (full library). Positive integrants were selected by treating with puromycin (hPGK-PuroR-P2A-HSV-TK) or flow sorting for mCherry (hPGK-HSV-TK-P2A-mCherry). Corresponding non-transduced cells for each cell line were used as positive control for puromycin selection. Lentiviruses for direct intratumoral delivery of HSV-TK interrupted by synMTERFD3i1-150-v6700 were 200X concentrated by ultracentrifugation (19,000g, 100 min at 4°C), resuspended in Opti-MEM (Gibco) and quantified using a qPCR Lentivirus Titer Kit (Applied Biological Materials).
North K., Benbarche S., Liu B., Pangallo J., Chen S., Stahl M., Bewersdorf J.P., Stanley R.F., Erickson C., Cho H., Pineda J.M., Thomas J.D., Polaski J.T., Belleville A.E., Gabel A.M., Udy D.B., Humbert O., Kiem H.P., Abdel-Wahab O, & Bradley R.K. (2022). Synthetic introns enable splicing factor mutation-dependent targeting of cancer cells. Nature biotechnology, 40(7), 1103-1113.
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9

Lentiviral Transduction of Human Claudin Genes

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The FG12 lentiviral vector (Addgene, Watertown, MA, USA) was used for gene transfection of human claudin-1, -2, -3, and -9 (hCLDNs) in HeLa and HepG2 cells. Each DNA fragment of human claudin was inserted into the FG12 vector. Twenty-four hours before gene transfection, HEK293T cells were seeded on a 6-well plate at a density of 0.95 × 105 cells/cm2. The transfection mixture of FG12/hCLDNs, the expression plasmid of the vesicular stomatitis virus (VSV-G), and the packaging plasmid psPAX2 were then added to the cells using Lipofectamine 3000 reagent. The spent culture medium was collected 72 h after transfection, centrifuged at 150 × g at 4 °C for 5 min, and the supernatant was stored at −80°C. Lentivirus titres were measured using a qPCR Lentivirus Titer Kit (Applied Biological Materials, Vancouver, Canada) according to the manufacturer’s instructions.
Arakawa H., Nakazono Y., Matsuoka N., Hayashi M., Shirasaka Y., Hirao A, & Tamai I. (2023). Induction of open-form bile canaliculus formation by hepatocytes for evaluation of biliary drug excretion. Communications Biology, 6, 866.
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10

Lentivirus Production for Gene Delivery

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Human embryonic kidney cells (HEK 293T, ATCC CRL-11268) cells were cultured in Dulbecco's modified eagle medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. All cell culture materials were purchased from Life Technologies (Carlsbad, CA, USA). Lentivirus was prepared using a previously established technique25 (link),36,37. Cells were co-transfected with vectors encoding plenti-CMV-Firefly luciferase, plenti-CMV-human SHH or plenti-CMV-human NT3 and the following packaging vectors: pMDL-GagPol, pRSV-Rev, pIVSVSV-G using Lipofectamine 2000 (Life Technologies). Supernatant was collected after 48 hours, concentrated in PEG-it (SystemBiosciences, Mountain View, CA) for 24 hours, precipitated using ultracentrifugation and resuspended in PBS. Titer was determined using a qPCR lentivirus titer kit (Applied Biological Materials, Inc., Richmond, BC, Canada).
Thomas A.M., Seidlits S.K., Goodman A.G., Kukushliev T.V., Hassani D.M., Cummings B.J., Anderson A.J, & Shea L.D. (2014). Sonic hedgehog and neurotrophin-3 increase oligodendrocyte numbers and myelination after spinal cord injury. Integrative biology : quantitative biosciences from nano to macro, 6(7), 694-705.
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