Gene segments of 1125, 943, 242, 95, and 48 bp located upstream of the major Transcription Start Site (TSS) of exon X1 were obtained by PCR and cloned in Firefly luciferase vector pGL4.15 (Promega, Fitchburg, WI, USA), using Q5 (New England Biolabs) or the high fidelity DNA polymerase Pfu (Stratagene, La Jolla, CA, USA) mixed with Takara Taq. In the same experiments, we used a 187 bp sequence upstream of the TRBV7.2 TSS, as a control promoter. In addition, a 400 bp fragment of the Enhancer ß (Eß) comprising the Eß‐core 7 was cloned in the unique BamH I site of pGL4.15 vectors containing the 943 bp fragment of the X1 promoter or the 187 bp‐long Vß7.2 promoter. The constructs were co‐transfected in triplicates in either HEK293T or Jurkat E6.1 cells with pGL4.75, a plasmid expressing the Renilla luciferase driven by a CMV promoter, as a transfection efficiency control. We used Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) and TransIT®Jurkat (Mirus Bio LLC) to transfect HEK293T and Jurkat cells, respectively. Luciferase activities were measured after 24–40 h using the Dual‐Glo® Luciferase Assay System (Promega) and a Glomax Discover plate reader (Promega). The Firefly luciferase activity was normalized to that of the Renilla luciferase and the results compared to those obtained with a promoterless vector, providing the ratios shown in Figure 3 A.
Transit jurkat
Manufactured by Mirus Bio
Sourced in United States
The TransIT-Jurkat is a transfection reagent designed for efficient delivery of DNA and RNA into Jurkat cells, a commonly used T cell line. It facilitates the introduction of genetic material into these cells for various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Fugene hd, by Promega (2 mentions)
Glomax discover plate reader, by Promega (1 mentions)
Dual glo luciferase assay system, by Promega (1 mentions)
35 mm coverslip dishes, by MatTek (1 mentions)
C2 ceramide, by Merck Group (1 mentions)
Phorbol 12 13 dibutyrate, by Merck Group (1 mentions)
C2 dihydroceramide, by Merck Group (1 mentions)
R geco1, by Addgene (1 mentions)
C6 ceramide, by Merck Group (1 mentions)
Thapsigargin, by Merck Group (1 mentions)
M csf, by Thermo Fisher Scientific (1 mentions)
11 protocols using transit jurkat
1
Cloning and Characterization of Promoter Regulatory Regions
2
Stable Cell Lines for GFP-CRACR2a Expression
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U2OS cells and HEK293T cells were cultured in DMEM containing 10% FBS and penicillin/streptomycin/l -glutamine. Jurkat cells were cultured in RPMI 1640 containing 10% FBS and penicillin/streptomycin/l -glutamine. Lentiviral transduction was used to create cell lines stably expressing GFP-CRACR2a, and GFP-positive cells were selected by FACS. Transient transfection of Jurkat cells was performed with TransIT-Jurkat (Mirus Bio) following the manufacturer’s protocol.
3
Transient Jurkat Cell Transfection
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A total of 2.0×10 6 cells were transfected with 2.5 µg of plasmid DNA and TransIT ® Jurkat (MirusBio) at a 1:5 ratio (mass/volume), according to the manufacturer's instructions. Next, cells were cultured for 24 h in Treg medium and taken into culture until analysis.
4
NLRP3 and TGR5 Regulation of IL-1β
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J774A.1 cells in 6-well plates were transfected with NLRP3, TGR5 small interfering RNA, or scrambled siRNA by using TransIT-Jurkat (Mirus Bio, Madison, WI, USA), followed by LPS stimulation and DCA treatment (100 μM, 24 h). IL-1β in supernatant was measured by ELISA. RNA oligonucleotides sequences were as follows: NLRP3, forward 5′-GGC GAG ACC UCU GGG AAA ATT-3′ and reverse 5′-UUU UCC CAG AGG UCU CGC CTT-3′; TGR5, forward 5′-CUG GAA CUC UGU UAU CGC UTT-3′ and reverse 5′-AGC GAU AAC AGA GUU CCA GTT-3′.
5
Modulation of Macrophage Signaling Pathways
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Macrophages were transfected with TGR5 small interfering RNA or scrambled siRNA in 6-well plates by using TransIT-Jurkat (Mirus Bio, Madison, WI), followed by DCA treatment (100 μM). TNF-α and NO levels in supernatant were measured accordingly. RNA oligonucleotides sequences were as follows: TGR5, forward 5ʹ-CUG GAA CUC UGU UAU CGC UTT-3ʹ and reverse 5ʹ-AGC GAU AAC AGA GUU CCA GTT-3ʹ. FXR, forward 5′-CCA AGA ACG CCG UGU ACA ATT-3′, reverse 5′-UUG UAC ACG GCG UUC UUG GTT-3′. S1PR2, forward 5′-CCU CUA CAA AGC CCA CUA UTT-3′, reverse 5′-AUA GUG GGC UUU GUA GAG GTT-3′. M2-mAchR, forward 5′-CCU CUA ACC UAC CCA GUU ATT-3′, reverse 5′-UAA CUG GGU AGG UUA GAG GTT-3′. TLR2, forward 5′-CCA AUC UCA CAA AUU UAC ATT-3′, reverse 5′-UGU AAA UUU GUG AGA UUG GTT-3′.
6
Mast Cell Transfection and Stimulation Protocol
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RBL-2H3 mast cells were maintained in monolayer culture through weekly passage as described previously (Gosse et al., 2005 (link)). For stimulation, cells were sensitized with 1 μg/ml anti-DNP IgE (Gosse et al., 2005 (link)) for 2-24 h. For transfection, cells were sparsely plated (1-3×105/ml) in six-well plates for fluorimetry experiments, or on #1.5 coverslips or in 35 mm cover slip dishes (MatTek Corporation, Ashland, USA) for confocal imaging. After overnight culture, cells were transfected using 2.5 μg DNA and 10 μl FuGENE HD (Promega) in 1 ml OptiMEM per well for 3-4 h in the presence of 1 ng/ml phorbol 12,13-dibutyrate to enhance DNA uptake for RBL cells (Gosse et al., 2005 (link)). Samples were then washed into full media and cultured for 16-24 h to allow for protein expression.
Jurkat T cells were maintained in suspension culture in RPMI medium with 10% fetal bovine serum and 1 μg/ml gentamicin. These cells were transfected using TransIT/Jurkat from Mirus Corporation for 16-24 h per manufacturer's recommendations.
Cells were then washed in buffered salt solution (BSS: 135 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 5.6 mM glucose, 20 mM HEPES, pH 7.4) with 1 mg/ml bovine serum albumin for experiments with short chain ceramides.
Jurkat T cells were maintained in suspension culture in RPMI medium with 10% fetal bovine serum and 1 μg/ml gentamicin. These cells were transfected using TransIT/Jurkat from Mirus Corporation for 16-24 h per manufacturer's recommendations.
Cells were then washed in buffered salt solution (BSS: 135 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 5.6 mM glucose, 20 mM HEPES, pH 7.4) with 1 mg/ml bovine serum albumin for experiments with short chain ceramides.
7
Calcium Signaling Assay Reagents
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Thapsigargin, phorbol 12,13-dibutyrate, C2-ceramide and C6-ceramide and C2-dihydroceramide were purchased from Sigma-Aldrich. FuGENE HD was from Promega (Madison, USA), and TransIT/Jurkat was from Mirus Corporation (Madison, USA). mAb OKT3 and mAb 4G10 were from Thermo Fisher Scientific and A488-conjugated goat anti-mouse γ2b was from Invitrogen. The genetically encoded Ca2+ indicator RGECO-1 (Zhao et al., 2011 (link)) was purchased from Addgene (plasmid #32444; Cambridge, USA). A488-CTxB was from Invitrogen and TX-100 Surfact-Amps was from Thermo Fisher Scientific.
8
Culturing and Transfecting RAW 264.7, 293T, and BMDM Cells
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RAW 264.7 cells and 293T cells were cultured in DMEM medium supplemented with 10% FBS. 293T cells were transfected by using Lipofectamine 2000 (Invitrogen, 11668019). BMDM were obtained as previously described56 (link) with modification. Briefly, cells were prepared by flushing the bone marrow from femurs and tibias and then maintained in DMEM medium containing 10% FBS and supplemented with 10 ng/ml M-CSF (Peprotech, 315–02). Four to 5 d later, adherent cells were dissociated and cultured in DMEM supplemented with 10% FBS and growth factor. RAW 264.7 and BMDM cells were transfected with TransIT-Jurkat (Mirus Bio, MIR2125), according to the manufacturer's instruction.
9
RAW 264.7 Cell Transfection Protocol
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RAW 264.7 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum. RAW 264.7 cells were transfected with TransIT-Jurkat (Mirus Bio, Madison, WI, USA), according to the manufacturer's instructions.
10
Silencing S1PR2 and Cathepsin B in J774A.1 Cells
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J774A.1 cells were transfected with S1PR2 and cathepsin B small interfering RNA or scrambled siRNA by using TransIT-Jurkat (Mirus Bio, Madison, WI, USA). Cells were then used to perform further experiment after 24 hours. RNA oligonucleotides sequences were as follows: S1PR2, forward 5′- CCU CUA CAA AGC CCA CUA UTT-3′ and reverse 5′- AUA GUG GGC UUU GUA GAG GTT-3′; cathepsin B, forward 5′- GCU GAA GAC CUG CUU ACU UTT-3′ and reverse 5′- AAG UAA GCA GGU CUU CAG CTT-3′.
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