Profection kit

Manufactured by Promega

The Profection kit is a laboratory equipment product designed for DNA transfection. It provides a reliable and efficient method for introducing genetic material into cells. The kit includes the necessary reagents and protocols to facilitate this process.

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Lab products found in correlation

Dual luciferase reporter assay system, by Promega (2 mentions) Chloroquine dihydrochloride, by Merck Group (2 mentions) Diethylaminoethyl dextran, by Merck Group (1 mentions) Pei max 40000, by Polysciences (1 mentions) Bright glo luciferase assay system, by Promega (1 mentions) Amaxa nucleofection system, by Lonza (1 mentions) Pgl3 basic, by Promega (1 mentions) Recombinant human il 2, by BD (1 mentions) Naive cd4 t cell isolation kit, by Miltenyi Biotec (1 mentions) Pilenti sirna gfp lentiviral vectors, by Applied Biological Materials (1 mentions) Opti mem, by Thermo Fisher Scientific (1 mentions) Lenti x concentrator, by Takara Bio (1 mentions) Lentix titration kit, by Takara Bio (1 mentions) Foetal calf serum, by Thermo Fisher Scientific (1 mentions) Gibco dmem, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

Profection mammalian transfection kit ProFection Calcium Phosphate Kit ProFection

10 protocols using profection kit

Tet2 and PPARγ Transcriptional Assay

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Reporter plasmids were co-transfected with expression vectors of Tet2-CD, Tet2-HxD, and PPARγ into 293T cells using a Profection kit (Promega Corporation). The Renilla reference plasmid was co-transfected to normalize transfection efficiency, and the mass of transfected plasmids was balanced with empty vector (pCDH-EGFP). 48 hr after transfection, cells were harvested and measured using the Dual-Luciferase Reporter Assay System (Promega Corporation).

Bian F., Xiang M., Sneha D.V., Dongjoo Y., Lauren R.C., Anushka P., Sarah F, & Sona K. (2018). TET2 facilitates PPARγ agonist–mediated gene regulation and insulin sensitization in adipocytes. Metabolism: clinical and experimental, 89, 39-47.

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HIV-1 Pseudovirus Neutralization Assay

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Virus neutralization was measured using HIV-1 pseudoviruses with TZM-bl target cells as described previously (20 (link)). HIV-1 pseudoviruses were produced in transfected 293T cells using a ProFection kit (Promega) or polyethylenimine (PEI) MAX40,000 (Polysciences). Prior to testing in the neutralization assay, sera were heat-inactivated (56°C for 30 min). Virus was incubated for 1 h at 37°C with serially diluted sera and then added to TZM-bl cells in the presence of diethylaminoethyl-dextran (Sigma). Virus infection was determined after 48 h using the Bright-Glo Luciferase Assay System (Promega). For peptide absorption assay, V3 peptide (40 μg/mL) was added to serum 1 h before the addition of virus.

Hioe C.E., Kumar R., Upadhyay C., Jan M., Fox A., Itri V., Peachman K.K., Rao M., Liu L., Lo N.C., Tuen M., Jiang X., Kong X.P, & Zolla-Pazner S. (2018). Modulation of Antibody Responses to the V1V2 and V3 Regions of HIV-1 Envelope by Immune Complex Vaccines. Frontiers in Immunology, 9, 2441.

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Regulation of Ucp1 Promoter Activity

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The mouse Ucp1 promoter was amplified and inserted into the KpnI and XhoI sites of pGL3-basic (Promega). HEK293T cells were cultured in 24-well plates and co-transfected with PGC-1α plasmid (0.2ug/well), IRF4 plasmid (0.2ug/well), luciferase reporter construct (0.2ug/well), and galactosidase expression vector (control reporter) (0.006 ug/well) with the Profection™ kit (Promega), according to the manufacturer’s protocol. The mass of transfected plasmids was balanced with empty vector (pCDH-EGFP). After 48h, cells were harvested and measured using the Dual-Luciferase Reporter assay system (Promega), and luciferase activity was divided by Renilla luciferase activity (control reporter) to normalize for transfection efficiency. Alternatively, SVFs were extracted from BAT and then six days after adipogenic stimulation, adipocytes were detached with trypsin and transfected using the Amaxa nucleofection system (Amaxa Biosystems). Transfections were performed with 1µg reporter construct along with 0.5µg IRF4-pCDH expression vector, 1 µg pcDNA- PGC-1α with 25 ng galactosidase expression vector. After 48 h, cells were harvested and measured using the Dual-Luciferase Reporter assay system (Promega). All luciferase assay experiments were performed 3 times at least, and each conducted in triplicate.

Kong X., Banks A., Liu T., Kazak L., Rao R.R., Cohen P., Wang X., Yu S., Lo J.C., Tseng Y.H., Cypess A.M., Xue R., Kleiner S., Kang S., Spiegelman B.M, & Rosen E.D. (2014). IRF4 is a key thermogenic transcriptional partner of PGC-1α. Cell, 158(1), 69-83.

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Lentiviral Transduction of Naive CD4+ T Cells

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Human RORC (RORγ) cDNA was synthesized, cloned into pUC57, and shuttled into an HIV-1–derived lentiviral (HDV) plasmid containing IRES-murine CD24 (HSA) downstream of multi-cloning site (cloning done at Genscript; plasmid gift of D. Unutmaz; Sundrud et al., 2003 (link)). VSV-G pseudotyped empty or RORC-containing HDV particles with were produced in HEK293T cells by calcium phosphate transfection according to the manufacturer’s protocol (Profection kit; Promega). Lentiviral supernatants were concentrated by centrifugation (Sundrud et al., 2009 (link)), resuspended in complete T cell medium, and stored at −80°C. For T cell transduction experiments, purified naive CD4⁺ T cells (isolated using naive CD4 T-Cell isolation Kit; Miltenyi Biotec) were activated with anti-CD3–/anti-CD28–coated beads and infected with lentiviral supernatants (MOI = 50). Viral supernatants were removed on day 3 and infected T cells were expanded in media containing 10 U/ml recombinant human IL-2 (BD).

Ramesh R., Kozhaya L., McKevitt K., Djuretic I.M., Carlson T.J., Quintero M.A., McCauley J.L., Abreu M.T., Unutmaz D, & Sundrud M.S. (2014). Pro-inflammatory human Th17 cells selectively express P-glycoprotein and are refractory to glucocorticoids. The Journal of Experimental Medicine, 211(1), 89-104.

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Lentiviral Production for siRNA Knockdown

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Lentiviruses were produced from sets of four mouse piLenti-siRNA-GFP lentiviral vectors (Applied Biological Materials, Inc. Richmond, BC, Canada) targeting Pelp1 (cat no. i029598), Ncoa2 (cat no. i033105), Ncoa1 (cat no. i038883), Ncoa6 (cat no. i042804), Ncor1 (cat no. i044954), or Ncor2 (cat no. i042611), or the Control piLenti-siRNA-GFP lentiviral vector (cat no. LV015-G). HEK-293T cells in 3× 10 cm dishes per construct, were co-transfected with 10 μg lentiviral vector, 7.5 μg psPAX2, and 2.5 μg pVSV-G per dish using ProFection^® kit (Promega) according to manufacturer’s protocol. At ~15 h post-transfection, the media was replaced with 2.5 ml fresh media. Conditioned media containing lentiviral particles (LVM) were collected every 12 h for another 48 h, pooled, and stored at 4 °C. LVM were passed through a 0.45-micron filter to remove cell debris, and then centrifuged at 4,000 RPM overnight to pellet the viruses. The pellets were resuspended in 50 μl sterile PBS and added with 8 μg/ml polybrene to day-1 C2C12 myoblasts.

Bruno N.E., Nwachukwu J.C., Srinivasan S., Nettles C.C., Izard T., Jin Z., Nowak J., Cameron M.D., Boregowda S.V., Phinney D.G., Elemento O., Liu X., Ortlund E.A., Houtman R., Stavreva D.A., Hager G.L., Kamenecka T.M., Kojetin D.J, & Nettles K.W. (2021). Chemical Systems Biology Reveals Mechanisms of Glucocorticoid Receptor Signaling. Nature chemical biology, 17(3), 307-316.

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Production and Titration of Recombinant Measles Viruses

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Vero-H2 and helper 293–3-46-H2 cell lines were previously described [20 (link)]. Recombinant MVs were produced according to previously published procedures [20 (link)]. In brief, helper 293–3-46-H2 cells were transfected using calcium phosphate precipitation (ProFection kit, Promega, E1200) with two plasmids encoding for the MV genome and MV polymerase (pEMCLa). Three days after transfection, the helper cells were overlaid on Vero-H2 cells. Appearance of infectious centers was monitored by observing green fluorescent protein (GFP) expression under fluorescence microscope. Single viruses were then picked and propagated on Vero-H2 cells.
For virus stock preparation, Vero-H2 cells were infected at a multiplicity of infection (MOI) of 0.05 in OptiMEM (Life Technologies, 221705) for 2 h at 37 °C. DMEM-10 medium was then added on top and transferred to 32 °C until 95% of the cells expressed GFP. Cell culture media were removed and cells were scraped in OptiMEM. Viral particles were released by two freeze–thaw cycles. Titers of virus stocks were determined by 50% end-point dilution (tissue culture infectious dose 50, or TCID₅₀) on Vero-H2 cells using the Spearman–Kärber method [30 ].

Wang Q., Vossen A., Ikeda Y, & Devaux P. (2019). Measles vector as a multigene delivery platform facilitating iPSC reprogramming. Gene therapy, 26(5), 151-164.

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Packaging and Transducing Cell Lines

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Phoenix A (amphotropic) cells were plated at a density of 4 × 10⁵ cells/well on a 6-well plate. On the following day, culture media was replaced with fresh media (Dulbecco’s modified Eagle medium, high glucose, 10% fetal bovine serum, pen-strep, L-glutamine). Chloroquine dihydrochloride (S764663; Sigma, St. Louis, MO) was added to Phoenix cells 5 minutes before transfection and 2 μg of plasmid DNA was transfected into cells using the Profection kit (E1200; Promega, Madison, WI). The media was changed 10 hours after transfection. Retroviral supernatant was collected the following day and 2 mL was added to GP+E86 cells, plated at 4 × 10⁴ cells/well on a 6-well dish the previous day, after filtration (0.45 μm) and supplemented with protamine sulfate (5 μg/mL). GP+E86 cells were transduced again on the following day. Viral supernatant from GP+E86 cells was collected on the following 2 days and used to transduce C3H10T1/2 cells.

Tsai E.A., Gilbert M.A., Grochowski C.M., Underkoffler L.A., Meng H., Zhang X., Wang M.M., Shitaye H., Hankenson K.D., Piccoli D., Lin H., Kamath B.M., Devoto M., Spinner N.B, & Loomes K.M. (2016). THBS2 Is a Candidate Modifier of Liver Disease Severity in Alagille Syndrome. Cellular and Molecular Gastroenterology and Hepatology, 2(5), 663-675.e2.

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Lentiviral Particle Production in HEK293T

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Lentiviral particles were produced in HEK293T cells as described in (Azzarelli et al., 2018a (link)). Briefly, HEK293T cells were transfected with third generation packaging vectors (vectors containing VSVG, gag-pol, rev and tat in a ratio 7:1:1.1) and with pBob-H2B-GFP vector (kind gift from Professor Rick Livesey), using Calcium Phosphate method (Promega ProFection^® kit, E1200). The morning after transfection, the medium was refreshed and then collected for viral production 36 h later. To concentrate the viral particles, the medium was mixed with the concentrator (Lenti-X™ Concentrator, Clontech, 63123) overnight at 4°C and then spun for 45 min at 2000× g at 4°C. After removing the supernatant, the pellet containing viral particles was resuspended in 200–300 µl of medium and stored in 20 µl aliquotes at −80°C. Viral titer was quantified using Titration kit (Lenti-X™ Titration kit, Clontech, 631235); the viral particles needed have been calculated using the formula: viral particle=[(total number of cells×MOI-multiplicity of infection)/titer)]×1000. The MOI used in our experiments is 10.

Azzarelli R., Ori M., Philpott A, & Simons B.D. (2021). Three-dimensional model of glioblastoma by co-culturing tumor stem cells with human brain organoids. Biology Open, 10(2), bio056416.

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Reverse Genetics Protocol for Moraten-eGFP Measles Virus

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Huh7.5 [22] (link), Vero [23] and 293T [24] (link) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM Gibco, Invitrogen) supplemented with 10% foetal calf serum (Gibco), 10 mM HEPES, 2 mM L-glutamine, 10 µg/ml gentamycine and 1% non-essential amino acids for Huh7.5 cells at 37°C and 5% CO₂.
Moraten-eGFP measles virus was recovered by reverse genetics as described by Radecke et al. [25] (link). The helper cell line 293-3-46 stably expressing T7 polymerase, MeV N and P proteins [25] (link) was transfected using the ProFection kit (Promega) with plasmids coding for MeV genome with an additional eGFP gene and MeV-L protein (pEMCLa). Three days after transfection, cells were overlaid on Vero cells. Upon appearance, isolated syncytia were picked and individually propagated on Vero cells. Virus stock was produced after a second passage at multiplicity of infection (MOI) 0.03 on Vero cells. Virus was checked for lack of mycoplasma contamination, sequence accuracy and infectivity (virus titration).

Louber J., Kowalinski E., Bloyet L.M., Brunel J., Cusack S, & Gerlier D. (2014). RIG-I Self-Oligomerization Is Either Dispensable or Very Transient for Signal Transduction. PLoS ONE, 9(9), e108770.

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Retroviral Transduction of C3H10T1/2 Cells

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Phoenix A (amphotropic) cells were plated at a density of 4 × 10⁵ cells/well on a 6-well plate. On the following day, culture media was replaced with fresh media (Dulbecco's modified Eagle medium, high glucose, 10% fetal bovine serum, pen-strep, L-glutamine). Chloroquine dihydrochloride (S764663; Sigma, St. Louis, MO) was added to Phoenix cells 5 minutes before transfection and 2 μg of plasmid DNA was transfected into cells using the Profection kit (E1200; Promega, Madison, WI). The media was changed 10 hours after transfection. Retroviral supernatant was collected the following day and 2 mL was added to GP+E86 cells, plated at 4 × 10⁴ cells/well on a 6-well dish the previous day, after filtration (0.45 μm) and supplemented with protamine sulfate (5 μg/mL). GP+E86 cells were transduced again on the following day. Viral supernatant from GP+E86 cells was collected on the following 2 days and used to transduce C3H10T1/2 cells.

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