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Hek blue il 12 cells

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HEK-Blue IL-12 cells are a reporter cell line derived from human embryonic kidney (HEK293) cells. These cells stably express the SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of an IL-12-inducible promoter. They can be used to monitor IL-12 activity.

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Lab products found in correlation

Quanti blue, by InvivoGen (4 mentions) Penicillin streptomycin glutamine (psg), by Thermo Fisher Scientific (2 mentions) Hek blue il 2 cells, by InvivoGen (1 mentions) Fetal bovine serum (fbs), by Avantor (1 mentions) Dulbecco s modified eagle s medium, by Corning (1 mentions) Ficoll paque, by Cytiva (1 mentions) Cytation 3 multi mode reader, by Agilent Technologies (1 mentions) Quanti blue solution, by InvivoGen (1 mentions) Normacin, by InvivoGen (1 mentions) Hek blue il 18 cells, by InvivoGen (1 mentions) Hek blue selection, by InvivoGen (1 mentions) Penicillin streptomycin, by GE Healthcare (1 mentions) Fetal bovine serum (fbs), by GE Healthcare (1 mentions)

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HEK-Blue cells (12 citations) HEK Blue IL-12 (3 citations) IL-12 (3 citations) Hek-Blue IL-12 reporter cells (2 citations)

8 protocols using hek blue il 12 cells

1

Assessing Activatable Cytokine Activity

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Example 20

HEK-Blue IL12 cells (InvivoGen) were plated in suspension at a concentration of 250,000 cells/well in culture media with or without 40 mg/ml human serum albumin (HSA) and stimulated with a dilution series of recombinant hIL12, chimeric IL12 (mouse p35/human p40) or activatable hIL12 for 24 hours at 37° C. and 5% CO2. Activity of uncleaved and cleaved activatable hIL12 was tested. Cleaved inducible hIL12 was generated by incubation with active MMP9. IL12 activity was assessed by quantification of Secreted Alkaline Phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen), a colorimetric based assay.

HEK-Blue IL2 cells (InvivoGen) were plated in suspension at a concentration of 50,000 cells/well in culture media with or without 15-40 mg/ml human serum albumin (HSA) and stimulated with a dilution series of recombinant hIL2 or activatable hIL2 for 24 hours at 37° C. and 5% CO2. Activity of uncleaved and cleaved activatable hIL2 was tested. Cleaved inducible hIL2 was generated by incubation with active MMP9 or another protease. IL2 activity was assessed by quantification of Secreted Alkaline Phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen), a colorimetric based assay. Results are shown in FIGS. 59-62.

US11739132B2. Separation moieties and methods of use thereof (2023-08-29). Werewolf Therapeutics, Inc. [US]. Inventors: William Winston [US], Luke Evnin [US], Vinay Bhaskar [US], Giselle Knudsen [US], Daniel J. Hicklin [US], Cynthia Seidel-Dugan [US], Jose Andres Salmeron Garcia [US], Heather R. Brodkin [US].
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2

Quantifying IL-12 Activity in HEK-Blue Cells

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Example 2

HEK-Blue IL-12 cells (InvivoGen) were plated in suspension at a concentration of 250,000 cells/well in culture media with or without 40 mg/ml human serum albumin (HSA) and stimulated with a dilution series of recombinant hIL-12, chimeric IL-12 (mouse p35/human p40) or activatable hIL-12 for 24 hours at 37° C. and 5% CO2. Activity of uncleaved and cleaved activatable hIL-12 was tested. Cleaved inducible hIL-12 was generated by incubation with active MMP9. IL-12 activity was assessed by quantification of Secreted Alkaline Phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen), a colorimetric based assay. Results are shown in FIGS. 7a, 7b, 8a-8f, and 11a-11d.

US11453710B2. Activatable interleukin 12 polypeptides and methods of use thereof (2022-09-27). Werewolf Therapeutics, Inc. [US]. Inventors: William Winston [US], Daniel Hicklin [US], Vinay Bhaskar [US], Luke Evnin [US], Patrick Baeuerle [DE], Jose Andres Salmeron Garcia [US], Heather Brodkin [US], Cynthia Seidel-Dugan [US].
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3

Evaluating IL-12 Bioactivity Using HEK-Blue™ Assay

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A HEK-Blue™ assay was used to prove IL-12 bioactivity in vitro. HEK-Blue™ IL-12 cells (InvivoGen, #hkb-il12) are designed to detect bioactive human and murine IL-12. To show in vitro bioactivity of IL-12 derived from AAV plasmids or AAV vectors, cells were cultured, transfected with plasmids or transduced with AAV vectors, and a reporter assay carried out according to manufacturer’s instructions. To show bioactivity of IL-12 expressed from the AAVs in vivo, plasma obtained from AAV-injected mice was examined in the HEK-Blue™ assay.
Düchs M.J., Kratzer R.F., Vieyra-Garcia P., Strobel B., Schönberger T., Groß P., Aljayyoussi G., Gupta A., Lang I., Klein H., Morilla S.M., Hopf S., Park J., Kreuz S., Klugmann M, & Igney F.H. (2024). Riboswitch-controlled IL-12 gene therapy reduces hepatocellular cancer in mice. Frontiers in Immunology, 15, 1360063.
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4

Quantifying IL-12 Activity in HEK-Blue Cells

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Example 2

HEK-Blue IL-12 cells (InvivoGen) were plated in suspension at a concentration of 250,000 cells/well in culture media with or without 40 mg/ml human serum albumin (HSA) and stimulated with a dilution series of recombinant hIL-12, chimeric IL-12 (mouse p35/human p40) or activatable hIL-12 for 24 hours at 37° C. and 5% CO2. Activity of uncleaved and cleaved activatable hIL-12 was tested. Cleaved inducible hIL-12 was generated by incubation with active MMP9. IL-12 activity was assessed by quantification of Secreted Alkaline Phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen), a colorimetric based assay. Results are shown in FIGS. 7a, 7b, 8a-8f, and 11a-11d.

US10696723B2. Activatable interleukin 12 polypeptides (2020-06-30). Werewolf Therapeutics, Inc. [US]. Inventors: William Winston [US], Daniel Hicklin [US], Vinay Bhaskar [US], Luke Evnin [US], Patrick Baeuerle [DE], Jose Andres Salmeron Garcia [US], Heather Brodkin [US], Cynthia Seidel-Dugan [US].
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5

HEK-Blue IL-12 Reporter Assay

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HEK-Blue IL-12 cells (InvivoGen) express a signal transducer and activator of transcription 4 (STAT4)–inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene that is triggered upon binding of IL-12 to IL-12R. HEK-Blue IL-12 cells were seeded onto a 96-well plate at 50,000 cells per well and exposed to medium containing mIL-12 or mIL-12Fc (0.2 to 100 ng/ml). After 18 hours, SEAP concentrations in supernatants were detected through the addition of Quanti-Blue (InvivoGen) and quantified via absorbance readings at 650 nm.
Sagiv-Barfi I., Czerwinski D.K., Shree T., Lohmeyer J.J, & Levy R. (2022). Intratumoral immunotherapy relies on B and T cell collaboration. Science immunology, 7(71), eabn5859.
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6

Culturing HEK-Blue IL-12 and NALM-6 cells

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HEK-Blue IL-12 cells (Invivogen)
were cultured according to the manufacturer’s recommendations
in Dulbecco’s modified Eagle’s medium (Corning) supplemented
with 10% heat-inactivated fetal bovine serum (FBS) (VWR) and 100 IU/mL
pen-strep (VWR). NALM-6 cells (gifted from Dr. Lia Gore, University
of Colorado) were cultured in RPMI 1640 supplemented with 10% FBS
and 100 U/mL penicillin and 100 μg/mL streptomycin. Primary
CD8+ T cells were obtained from normal donor human buffy coats (LifeSouth)
via Ficoll-Paque (Cytiva) gradient selection for peripheral blood
mononuclear cells followed by human CD8+ T cell negative magnetic
selection (Stem Cell Technologies). Primary CD8+ T cells were thawed
in supplemented RPMI 1640 2–12 h prior to the start of experiments.
Birnbaum L.A., Sullivan E.C., Do P., Uricoli B., Raikar S.S., Porter C.C., Henry C.J, & Dreaden E.C. (2023). Multicolor Light-Induced Immune Activation via Polymer Photocaged Cytokines. Biomacromolecules, 24(3), 1164-1172.
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7

Quantifying IL-12 Bioactivity Using HEK-Blue Cells

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HEK-Blue IL-12 cells (InvivoGen), designed to detect bioactive human or mouse IL-12, were used to quantify the functionality of plasmid-derived IL-12 produced by the pIL12 and pIL12-CXCL9 plasmids. The bioassay was performed as described by the manufacturer, using a 2-fold dilution series of IL-12 ranging from 0 to 25.6 ng/mL. Briefly, HEK-Blue IL-12 cells at 50,000 cells/180 μL/well were incubated with 20 μL of IL-12 for 14–16 h at 37°C and 5% CO2. Afterward, 20 μL of the resulting conditioned media were added to 180 μL of freshly made QUANTI-Blue Solution (InvivoGen). Colorimetric assay was incubated for 30 min at 37°C and analyzed with a Cytation 3 Multi-Mode Reader (BioTek, Winooski, VT, USA) measuring absorbance at 630 nm.
Lee J.Y., Nguyen B., Mukhopadhyay A., Han M., Zhang J., Gujar R., Salazar J., Hermiz R., Svenson L., Browning E., Lyerly H.K., Canton D.A., Fisher D., Daud A., Algazi A., Skitzki J, & Twitty C.G. (2022). Amplification of the CXCR3/CXCL9 axis via intratumoral electroporation of plasmid CXCL9 synergizes with plasmid IL-12 therapy to elicit robust anti-tumor immunity. Molecular Therapy Oncolytics, 25, 174-188.
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8

Cell Culture Protocols for K562, HEK-Blue, and 32Dβ Lines

Cited 1 time
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K562 cells (ATCC, CCL-243) were obtained from ATCC in 2008, viably cryopreserved, thawed for use in these studies, and maintained for no more than 2 months at a time in continuous culture as described.24 (link) Prior to our studies, the K562 cells were authenticated by confirming cell growth morphology (lymphoblast), growth characteristics, and functionally as NK-cell-sensitive targets in 2014 and 2015. Cells were cultured in RPMI 1640 supplemented with L-glutamine, HEPES, non-essential amino acid (NEAA), sodium pyruvate, and penicillin/streptomycin containing 10% fetal bovine serum (FBS) (Hyclone/GE Healthcare, Logan, UT).
HEK-Blue IL-18 cells (IL-18 sensor cells) and HEK-Blue IL-12 cells (IL-12 sensor cells) from InvivoGen were cultured in complete HEK-Blue media (I10 media), consisting of IMDM, 10% FBS (HyClone/GE Healthcare), 1X penicillin-streptomycin-glutamine (Thermo Fisher Scientific, Dallas, TX); 100 μg/mL normacin, and 1X HEK-Blue selection (InvivoGen). The 32D-IL-2/15Rβ (32Dβ) cells were constructed at Altor BioScience, cultured in complete 32Dβ media containing IMDM-10 media plus 25 ng/mL rhIL-2, and maintained at a cell density between 1.5 × 104 - 2 × 106 cells/mL at 37°C and 5% CO2.
Cubitt C.C., McClain E., Becker-Hapak M., Foltz J.A., Wong P., Wagner J.A., Neal C.C., Marin N.D., Marsala L., Foster M., Schappe T., Soon-Shiong P., Lee J., Berrien-Elliott M.M, & Fehniger T.A. (2022). A novel fusion protein scaffold 18/12/TxM activates the IL-12, IL-15, and IL-18 receptors to induce human memory-like natural killer cells. Molecular Therapy Oncolytics, 24, 585-596.
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