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Jurkat lucia nfat

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The Jurkat-Lucia™ NFAT is a reporter cell line designed for the monitoring of NFAT (Nuclear Factor of Activated T cells) activation. This cell line constitutively expresses the Lucia luciferase reporter gene under the control of an NFAT-inducible promoter, allowing for the quantitative measurement of NFAT activation in response to various stimuli.

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Lab products found in correlation

Quanti luc gold substrate, by InvivoGen (1 mentions) Anti human cd107a antibody, by BD (1 mentions) Jurkat lucia nfat cd16, by InvivoGen (1 mentions) Spectramax plate reader, by Molecular Devices (1 mentions)

Spelling variants of this product

Jurkat Lucia NFAT CD16 cells (14 citations) Jurkat-Lucia™ NFAT-CD32 (2 citations) Jurkat-Lucia™ NFAT-CD16 (2 citations) Jurkat-Lucia NFAT cells (2 citations)

4 protocols using jurkat lucia nfat

1

Jurkat CAR-T Cell Activation and Degranulation

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Reporter cell line Jurkat-NFAT-Lucia (Invivogen, France) expressing CD19-CAR (Jurkat CD19-CAR) or IL13-CAR (Jurkat IL13-CAR) were obtained by lentiviral transduction as described before. For activation assay 5x10 4 Jurkat CAR-T cells were mixed with freshly isolated antigen-positive or antigen-negative AVs at CAR-T cell-to-AVs cell ratios (0, 4:1, 2:1, 1:1, 1:2, 1:5, 1:10 or 1:20) in a 96-well plate and incubated for 24 hours at 37°C. 25µl of supernatant was separated from cells by centrifugation (4°C, 300g, 5 min) and transferred to the opaque 96-well plate. Activation of reporter Jurkat-NFAT-Lucia CAR-T cells was measured by level of luciferase activity following reaction with Quanti-luc Gold substrate (Invivogen, France) according to the manufacturer's instructions.
T cell degranulation assay: IL13-CAR T cells were resuspended in TexMACS media in concentration 2x10 6 cells/ml. 100 µl of cell suspension was transferred to the 96-well plate and 2µl of anti-human CD107a antibody (BD) were added in each well. CAR-T cells were mixed with either antigen-positive or antigen-negative AVs at ratio 1:1, or kept untreated (ctl) and were incubated for 2 hours on 4°C. Cell suspension was washed twice with TexMACS media (4°C, 300g, 5 min), stained with anti-human CD3/CD4/CD8 antibodies and analyzed by flow cytometry.
Ukrainskaya V., Rubtsov Y., Pershin D., Podoplelova N., Terekhov S., Yaroshevich I., Sokolova A., Bagrov D., Kulakovskaya E., Shipunova V., Deyev S., Ziganshin R., Chernov A., Telegin G., Maksimov E., Markov O., Oshchepkova A., Zenkova M., Xie J., Zhang H., Gabibov A., Maschan M., Stepanov A, & Lerner R. (2021). Antigen-Specific Stimulation and Expansion of CAR-T Cells Using Membrane Vesicles as Target Cell Surrogates. Small (Weinheim an der Bergstrasse, Germany), 17(45).
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2

Generation of NFAT-reporter Jurkat Cells with FcR Expression

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The Jurkat-Lucia™ NFAT and Jurkat-Lucia™ NFAT-CD16 (hereafter designated Jurkat-NFAT-hCD16) cells were purchased from InvivoGen. Both cells were derived from the human Jurkat cell line by stable integration of an NFAT-inducible Lucia luciferase reporter (InvivoGen). The former is the parental reporter cell line and the latter having additional expressing of human Fc receptor CD16A (FcγRIIIA; V158 allotype). For the generation of Jurkat-NFAT-hCD32 cells, the parental Jurkat-NFAT cells were stably transduced with a lentiviral vectored human CD32A (FcγRIIA; H131 allotype) expression cassette. The CD32A cDNA is linked with an IRES-mRuby3-P2A-BsR cassette (synthesized by Generalbiol, Anhui, China) and cloned into the pLV-EF1α-MCS-IRES-Bsd vector. The presence of IRES-driven expression of mRuby3 and Bsd enable convenient positive-selections via blasticidin supplement and FACS-sorting. The Jurkat-NFAT-hCD16 and Jurkat-NFAT-hCD32 cells were cultured in SuperCulture serum-free lymphocyte medium (DAKEWEI) with Normocin (10 μg/mL), Blasticidin (10 μg/mL), and Zeocin (10 μg/mL) at 37 ℃ in a stackable CO2 (8%) incubator shaker.
Hong Y., Guo H., Wei M., Zhang Y., Fang M., Cheng T., Li Z., Ge S., Yao X., Yuan Q, & Xia N. (2022). Cell-based reporter assays for measurements of antibody-mediated cellular cytotoxicity and phagocytosis against SARS-CoV-2 spike protein. Journal of Virological Methods, 307, 114564.
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3

T-cell Activation Assay with Bispecific Antibody

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MEC1 or MEC1-002 cells (2×105 cells) were seeded in 96-well round-bottom plates, and T-biAb or Dulbecco’s phosphate-buffered saline (DPBS; vehicle) was added followed by a 30 min incubation at 37°C. Jurkat cells engineered to express luciferase under the transcriptional control of NFAT (Jurkat-Lucia NFAT, InvivoGen) were added at an effector-to-target (E:T) ratio of 1:1 (2×105 cells). After overnight culture, 20 µL of supernatant was combined with 50 µL of QUANTI-Luc substrate, and luminescence was recorded after 15 min using a SpectraMax M5 plate reader. The positive control for NFAT activation consisted of Jurkat-Lucia NFAT cells plated alone with 0.5 mg/mL concanavalin A. Percent NFAT activation was calculated as follows:
NFATactivation(%)=T−biAbtreated−vehicleConA−vehicle∗100
Cyr M.G., Mhibik M., Qi J., Peng H., Chang J., Gaglione E.M., Eik D., Herrick J., Venables T., Novick S.J., Courouble V.V., Griffin P.R., Wiestner A, & Rader C. (2022). Patient-derived Siglec-6-targeting antibodies engineered for T-cell recruitment have potential therapeutic utility in chronic lymphocytic leukemia. Journal for Immunotherapy of Cancer, 10(11), e004850.
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4

CD16 Reporter Assay for ADCC Surrogate

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A surrogate for antibody-mediated cellular cytotoxicity was measured using a CD16 reporter assay system used previously (78 (link)). Jurkat Lucia NFAT (Invivogen, jktl-nfat-cd16) cells were cultured according to manufacturer’s instructions. Cultured cells express CD16 (FcγRIIIa) which when engaged on the cell surface leads to luciferase secretion from the cell. First, high binding 96-well plates were coated overnight at 4°C with 1 μg/mL of spike or RBD antigen. Following incubation, plates were washed (PBS + 0.1% Tween20) and blocked (PBS + 2.5% BSA) at room temperature for 1 hr. Following plate washing, 100,000 cells per well and dilute serum samples were added to each well in cell culture media lacking antibiotics in a 200 μl volume. Following 24 hr incubation, 25 μL of supernatant from each well was transferred into a white 96 well plate which 75 μL of quantiluc substrate was immediately added. Following 10 min incubation, plates were read on a SpectraMax plate reader (Molecular Devices). VRC01 was used as a negative control; cell stimulation cocktail (Therm, 00-4970-93) and ionomycin and S309 served as positive controls. All samples were run in three biological replicates.
Hederman A.P., Natarajan H., Wiener J.A., Wright P.F., Bloch E.M., Tobian A.A., Redd A.D., Blankson J.N., Rottenstreich A., Zarbiv G., Wolf D., Goetghebuer T., Marchant A, & Ackerman M.E. (2022). SARS-CoV-2 mRNA vaccination elicits broad and potent Fc effector functions to VOCs in vulnerable populations. medRxiv.
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