Hek 293 blue cells

Manufactured by InvivoGen

HEK 293 Blue Cells are a genetically engineered cell line derived from human embryonic kidney (HEK) 293 cells. These cells express a blue fluorescent protein, allowing for easy visualization and tracking in cell-based assays.

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Lab products found in correlation

Quanti blue, by InvivoGen (2 mentions) Gentamicin, by Thermo Fisher Scientific (1 mentions) Odn2395, by InvivoGen (1 mentions)

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HEK-Blue cells (12 citations)

6 protocols using hek 293 blue cells

Annexin II Peptide Activation of TLR2 and TLR4

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Example 5

TLR2-transfected HEK 293 Blue Cells and TLR4-transfected HEK 293 Blue Cells (Invivogen, San Diego, Calif.) were pulsed with the N-terminal 15 amino acid annexin II fragment peptide (15aa Peptide, SEQ ID NO:5) at different concentrations. Cells were incubated for 48 hours. Following incubation, supernatant was added to Quanti-Blue (Invivogen, San Diego, Calif.) for secreted alkaline phosphatase detection. Results are shown in FIG. 6.

TLR2-transfected HEK 293 Blue Cells and TLR4-transfected HEK 293 Blue Cells (Invivogen, San Diego, Calif.) were pulsed with the N-terminal 15 amino acid annexin II fragment peptide (15aa Peptide, SEQ ID NO:5) or one of the annexin II variants shown in Table 1. Cells were incubated for 48 hours. Following incubation, supernatant was added to Quanti-Blue (Invivogen, San Diego, Calif.) for secreted alkaline phosphatase detection.

US10206986B2. Annexin II variant compositions and methods (2019-02-19). REGENTS OF THE UNIVERSITY OF MINNESOTA [US]. Inventors: John R. Ohlfest [US], Michael R. Olin [US].

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Purification and Labeling of Mouse Leptin

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Mouse leptin was produced and purified as described earlier [26 (link)] and 4.10-mAlb by the VIB Protein Service Facility up to 95 % purity. LPS contaminations were less than 1 EU per mg protein. LPS content was measured using the limulus amebocyte lysate in combination with a chromogenic substrate (Cambrex), or with the Toll-like receptor 4 expressing Hek293-BlueTM cells (InvivoGen) according to the manufacturer’s instructions. Antibodies Alexafluor labelled anti-CD4 and PE labelled anti-CD8 (both from eBiosciences) were used according to the manufacturer’s instructions.

Zabeau L., Jensen C.J., Seeuws S., Venken K., Verhee A., Catteeuw D., van Loo G., Chen H., Walder K., Hollis J., Foote S., Morris M.J., Van der Heyden J., Peelman F., Oldfield B.J., Rubio J.P., Elewaut D, & Tavernier J. (2014). Leptin’s metabolic and immune functions can be uncoupled at the ligand/receptor interaction level. Cellular and Molecular Life Sciences, 72(3), 629-644.

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Cell Culture and Transfection Protocol

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Hek293T, Hek293-BlueTM cells (InvivoGen) and MCF7 cells were cultured in 10 % CO₂ humidified atmosphere at 37 °C and grown using DMEM with 4,500 mg/l glucose, 10 % foetal bovine serum and 50 μg/ml gentamicin (all from Invitrogen). Hek293T cells were transfected using a standard calcium phosphate precipitation procedure [22 (link)].

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TLR9-Dependent NFκB Activation Assay

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For TLR9-dependent NFκB reporter assay, HEK293-Blue cells were used per as the manufacturer’s protocol (Invivogen, San Diego, CA). Cells were generated by co-transfecting the murine TLR9 gene and an inducible SEAP reporter gene into HEK293 cells. The SEAP gene was placed under the control of the interferon-beta (IFNβ) minimal promoter fused to five NFκB and activator protein-1 (AP-1) binding sites. Stimulation with a TLR9 ligand activates NFκB and AP-1, which induce the production of SEAP and are measured by a plate reader at 650 nm. Cells were treated with ODN2395 and SD101 at increasing doses (0.004–10 µM) for 21 h. As a negative sequence control for ODN2395 ODN5328 (C) (Invivogen, San Diego, CA) was used. Sequence control contains GpC dinucleotides instead of CpG present in ODN2395 (Invivogen, San Diego, CA).

Ghosh C.C., Heatherton K.R., Connell K.P., Alexander I.S., Greer D.A., LaPorte J., Guha P., Cox B.F, & Katz S.C. (2022). Regional infusion of a class C TLR9 agonist enhances liver tumor microenvironment reprogramming and MDSC reduction to improve responsiveness to systemic checkpoint inhibition. Cancer Gene Therapy, 29(12), 1854-1865.

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Isolation and Stimulation of Human PBMCs

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Human PBMCs were isolated as described (Schlee et al., 2009 (link)). For stimulation, 4 × 10⁶ cells (PBMCs) were cultured in 96-well plates. The PBMC studies were approved by the local ethics committee (Ethikkommission der Medizinischen Fakultät Bonn) according to the ICH-GCP guidelines. Written informed consent was provided by voluntary blood donors.
To inhibit TLR7/8 activity, cells were pre-incubated with 2.5 μg/ml chloroquine for 30 min. Cells were kept in RPMI 1640 (10% FCS, 1.5 mM L-Glu, 100 U/ml penicilin, 100 μg/ml streptomycin). For transfection, nucleic acids were complexed with Lipofectamine(LF) 2000 (Life Technologies). HEK293^blue cells (Invivogen) are reporter cells for type 1 IFN. By incident, as demonstrated (Figures 2A and 2D), they show negligible RIG-I background activity. Murine BM-DCs were generated by culturing murine bone marrow cells for 7 days with GM-CSF.

Schuberth-Wagner C., Ludwig J., Bruder A.K., Herzner A.M., Zillinger T., Goldeck M., Schmidt T., Schmid-Burgk J.L., Kerber R., Wolter S., Stümpel J.P., Roth A., Bartok E., Drosten C., Coch C., Hornung V., Barchet W., Kümmerer B.M., Hartmann G, & Schlee M. (2015). A Conserved Histidine in the RNA Sensor RIG-I Controls Immune Tolerance to N1-2′O-Methylated Self RNA. Immunity, 43(1), 41-51.

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Annexin II-OVA Fusion Protein Stimulation

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Example 2

HEK 293 Blue Cells (Invivogen, San Diego, Calif.) stably transfected with hTLR2 were pulsed with annexin II-OVA fusion protein (A2OVA, SEQ ID NO:32) at equal concentration as annexin II monomer (A2 Peptide, SEQ ID NO:28). Scrambled annexin II N-terminus polypeptide (SEQ ID NO:29, Scrambled) and an annexin II fragment (SEQ ID NO:30, Minus p11) were used as controls. Cells were incubated for 48 hours. Following incubation, supernatant was added to Quanti-Blue (Invivogen, San Diego, Calif.) for secreted alkaline phosphatase detection. Results are shown in FIG. 3.

US10206986B2. Annexin II variant compositions and methods (2019-02-19). REGENTS OF THE UNIVERSITY OF MINNESOTA [US]. Inventors: John R. Ohlfest [US], Michael R. Olin [US].

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