Recombinant FcγRs ectodomains were purchased from Sino Biologic Inc. (North Wales, PA, USA). The pComb3X was kindly provided by Dennis Burton (Scripps Research Institute, La Jolla, CA, USA) and the pSecTag2 B was purchased from Invitrogen. IgG1 m336 and m912-mFc were produced in our group. The following antibodies were purchased: mouse anti-CD16A IgG1, 3G8 (Abcam, Cambridge, MA, USA); phycoerythrin (PE)-conjugate mouse anti-CD16A, PE conjugated mouse anti-FLAG (Miltenyi, Bergisch Gladbach, Germany); fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD64 (FcγRI) and CD32 (FcγRII) (Invitrogen); horseradish peroxidase (HRP) anti-M13 polyclonal (Pharmacia, Piscataway, NJ); HRP-conjugated mouse anti-FLAG tag, HRP conjugated goat anti-mouse IgG and HRP-conjugated goat anti-human IgG (Fc-specific) (Sigma-Aldrich). U937 cell was a gift from Anu Puri (National Cancer Institute, Frederick, MD). The following cell lines were purchased: Jurkat T (ATCC); 293 freestyle (Invitrogen) and Jurkat T over-expressing CD16A (Promega). Human blood was obtained from the NIH blood center.
Jurkat t
Manufactured by American Type Culture Collection
Sourced in United States
The Jurkat T cell line is a T lymphocyte cell line derived from a human T cell leukemia. It is a widely used model for the study of T cell signaling and activation.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (4 mentions)
Hek293t, by American Type Culture Collection (3 mentions)
Nivolumab, by Selleck Chemicals (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Ifn γ, by Thermo Fisher Scientific (2 mentions)
Azd4547, by Selleck Chemicals (2 mentions)
Fibroblast growth factor 2 (fgf2), by Thermo Fisher Scientific (2 mentions)
Jurkat t cells, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Welgene (2 mentions)
Thp 1, by American Type Culture Collection (2 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions)
Negative selection, by Miltenyi Biotec (2 mentions)
Pan t cell, by Miltenyi Biotec (2 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (2 mentions)
Fetal calf serum (fcs), by Gemini Bio (2 mentions)
Hek293t cell, by American Type Culture Collection (2 mentions)
Psectag2b, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated goat anti human igg fc specific, by Merck Group (1 mentions)
11 protocols using jurkat t
1
Recombinant FcγR Ectodomains Evaluation
2
Cell Culture and Reagent Preparation
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Cell culture and reagents H520, HCC95 and Jurkat T cells were purchased from ATCC and cultured using RPMI 1640 (HyClone, USA) with 10% fetal bovine serum (Gibco, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco, USA). LLC cells were purchased from ATCC and cultured using DMEM (HyClone, USA) with 10% fetal bovine serum (Gibco, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco, USA).
FGF2 and IFN-γ were purchased from PeproTech. AZD4547 and nivolumab were purchased from Selleck. AZD4547 was dissolved in DMSO, aliquoted, and stored as a Aldrich, USA) was stored at a concentration of 10 mM in DMSO at -20 °C. All cells were maintained in a humidified incubator at 37°C with 5% carbon dioxide.
FGF2 and IFN-γ were purchased from PeproTech. AZD4547 and nivolumab were purchased from Selleck. AZD4547 was dissolved in DMSO, aliquoted, and stored as a Aldrich, USA) was stored at a concentration of 10 mM in DMSO at -20 °C. All cells were maintained in a humidified incubator at 37°C with 5% carbon dioxide.
3
Cell Culture and Reagent Preparation
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Cell culture and reagents H520, HCC95 and Jurkat T cells were purchased from ATCC and cultured using RPMI 1640 (HyClone, USA) with 10% fetal bovine serum (Gibco, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco, USA). LLC cells were purchased from ATCC and cultured using DMEM (HyClone, USA) with 10% fetal bovine serum (Gibco, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco, USA).
FGF2 and IFN-γ were purchased from PeproTech. AZD4547 and nivolumab were purchased from Selleck. AZD4547 was dissolved in DMSO, aliquoted, and stored as a Aldrich, USA) was stored at a concentration of 10 mM in DMSO at -20 °C. All cells were maintained in a humidified incubator at 37°C with 5% carbon dioxide.
FGF2 and IFN-γ were purchased from PeproTech. AZD4547 and nivolumab were purchased from Selleck. AZD4547 was dissolved in DMSO, aliquoted, and stored as a Aldrich, USA) was stored at a concentration of 10 mM in DMSO at -20 °C. All cells were maintained in a humidified incubator at 37°C with 5% carbon dioxide.
4
Culture and Maintenance of HEK293T and Jurkat Cells
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Human embryonic kidney 293 T (HEK293T) and Jurkat T cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). HEK293T cells were cultured at 37°C and 10% CO2 in Dulbecco's modified Eagle's medium (DMEM; Welgene, Gyeongsan, Korea) supplemented with 10% fetal bovine serum (FBS; Welgene) and 1% penicillin/streptomycin (P/S; GE Healthcare, Chicago, IL, USA). Jurkat T cells were cultured in RPMI1640 (Gibco; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS and 1% P/S at 37°C in a 5% CO2 incubator.
5
Culturing Jurkat T, 293 T, and PBMC
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Jurkat T and 293 T-cell lines were purchased from American Type Culture Collection (Manassas, VA, USA). Human primary peripheral blood mononuclear cells (PBMC) from healthy volunteers were isolated from whole blood samples with Ficoll-Paque Plus (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) using density gradient separation. This study was approved by the Ethics Committee of Shanghai Changhai Hospital. Whole blood for the study was obtained from healthy volunteers who had provided written informed consent. Jurkat T cells and PBMC were cultured in RPMI 1640 growth medium supplemented with penicillin (100 U/ml), streptomycin (100 μg/ml), l -glutamine (2 mM), and 10% fetal bovine serum (FBS). 293 T cells were cultured in DMEM medium supplemented with penicillin (100 U/ml), streptomycin (100 μg/ml), and 10% FBS (All from Life Technologies GmbH, Darmstadt, Germany).
6
Cell Line Maintenance Protocol
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The human prostate cancer (LNCaP) cell line was purchased from the Korean Cell Line Bank (Seoul, Korea). Another human prostate cancer (DU145) and normal human dermal fibroblast cell lines were kindly provided by the German Collection of Microorganisms (DSMZ, Braunschweig, Germany) and Modern Cell & Tissue Technologies (Seoul, Korea). The normal prostate epithelial PNT2, Jurkat-T, NK92 and THP-1 were purchased from American Type Culture Collection (Manassas, VA). DU145 cells was maintained in Dulbecco’s Modified Eagles medium (Capricorn scientific, Ebsdorfergrund, Germany). LNCaP, PNT2, Jurkat-T, NK-92, THP-1 and normal human dermal fibroblast cells were maintained in RPMI-1640 medium (Life Technologies, Carlsbad, CA, USA). All the cells were maintained in the medium supplemented with 10% heat-inactivated fetal bovine serum (FBS, Life Technologies, Carlsbad, CA, USA) and 1% penicillin/streptomycin solution (Life Technologies, Carlsbad, CA, USA) at 37 °C with 5% CO2 in a humidified incubator.
7
Cell Line Culture Conditions
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Human cell lines 293T, A549, RKO, HeLa, KATOIII, AGS, MCF-7 and PANC-1, THP-1 and Jurkat T cells were from American Type Culture Collection (ATCC, Manassas, VA) while SNU-C5 cells were from the Korean Cell Line Bank (Seoul, Korea). SNU-C5, THP-1 and Jurkat T cells were cultured in RPMI1640 containing 10% fetal bovine serum (HyClone, Logan, UT) while all other cells were cultured in Dulbecco's Modified Eagle Medium (HyClone) containing 10% fetal bovine serum at 37°C under 5% CO2.
8
Cell Line Generation and Validation
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The human thyroid cancer cell line 8505C was obtained from Sigma in 2018; HEK 293T and Jurkat T cell lines were purchased from the American Type Culture Collection (ATCC) in 2019. 8505C and Jurkat T cell lines were cultured in RPMI-1640 (Corning) supplemented with 10% heat-inactivated fetal bovine serum (FBS); HEK 293T cell line was cultured in Dulbecco's Modified Eagle's Medium (DMEM, Corning) supplemented with 10% FBS. 8505C and Jurkat T cell lines were stably transduced with a Firefly Luciferase-F2A-GFP (FLuc-GFP) lentivirus (Biosettia) for in vivo assessment of tumor burden in mouse models. All cell lines were maintained at 37 °C in a 5% CO2 atmosphere and were routinely validated to be Mycoplasma free using a MycoAlert™ detection kit (Lonza).
9
Cell culture and T cell activation
10
Cell culture and T cell activation
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HEK293T cells (human embryonic kidney cells expressing SV40 large T antigen, negative for mycoplasma, originally obtained from the American Type Culture Collection) and skin fibroblast cells derived from TNFAIP3-deficient patients or normal donors were grown in Dulbecco’s modified Eagle’s medium (Life Technologies) plus 10% fetal calf serum and 1x antibiotics (Life Technologies). Jurkat-T (human leukaemic T lymphoma cell line, negative for mycoplasma, originally obtained from the American Type Culture Collection) cells and human PBMCs were grown in RPMI1640 medium (Life Technologies), plus 10% fetal calf serum (Gemini Bio-Products) and antibiotics. For intracellular TNF staining, pan-T cells purified from PBMCs by negative selection (Miltenyi) were cultured in complete RPMI 1640 in the presence of plate-bound anti-CD3 and soluble anti-CD28 (both 1 μg/mL) for 5 days. For IL-9 staining, rhIL-4 (30 ng/mL), TGF-β (5 ng/mL), IL-1β (10 ng/ml), and IL-2 (10 U/ml), together with anti-IFNγ (10 μg/mL) were added to total PBMCs, and cells were cultured for 48 hours under the same conditions.
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