Clonal populations of HeLa cell lines stably expressing human melanopsin (hOPN4) were created by lentiviral transduction under the CMV promoter. For transcriptional activation assays, cells were transfected with NFAT-luciferase reporter plasmid pGL4.30[luc2P/NFAT-RE/Hygro] (Promega E8481) using TransIT-LT1 transfection agent (Mirus Biotech) according to manufacturer protocols. Cells were plated on 1 cm coverslips for calcium imaging or black 24-well plates for gene expression assays, with collagen adhesion layers.
Luc2p nfat re hygro
Manufactured by Promega
Sourced in United States
The Luc2P/NFAT-RE/Hygro is a reporter construct that contains the Luc2P luciferase gene under the control of the NFAT response element. This reporter system can be used to monitor the activation of the NFAT signaling pathway in cells.
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Lab products found in correlation
One glo luciferase assay system, by Promega (1 mentions)
Acumen ex3, by SPT Labtech (1 mentions)
Dynabeads human t activator cd3 cd28, by Thermo Fisher Scientific (1 mentions)
Pef1α mcherry n1, by Takara Bio (1 mentions)
In fusion hd cloning kit, by Takara Bio (1 mentions)
Wallac arvo sx 1420 multilabel counter, by PerkinElmer (1 mentions)
Phrl tk vector, by Promega (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
4 protocols using luc2p nfat re hygro
1
Melanopsin Expression in HeLa Cells
2
Quantifying NF-kB and NFAT Activation
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NF-kB/Jurkat/GFP and stably transfected NF-kB/Jurkat/GFP/TIM-3 cells were plated at 2.5e4 cells/well in 96-well Microtest plates (BD Falcon) in RPMI +Glutamax Phenol Red -Free media (Life Technologies). Cells were stimulated overnight at 37°C with either Cell Stimulation Cocktail (CSC) containing phorbol myristate acetate (PMA) and ionomycin (eBioscience, San Diego, CA) or Human T-Activator CD3/CD28 Dynabeads at a ratio of 10:1 (beads:cells) (Life Technologies) and NF-κB reporter activity was assessed by imaging on the Acumen eX3 (TTP Labtech, Cambridge MA). To determine NFAT activity, cells were transiently transfected with 2μg of pGL4.30 (Luc2P/NFAT-RE/Hygro) (Promega, Madison, WI) per 106 cells and allowed to recover for 72h. Cells were stimulated as described and luciferase activity was assessed using the One-Glo luciferase assay system (Promega) and measured on an Envision (Perkin Elmer, Waltham, MA).
3
Generation of Optogenetic Tools for Neuroscience
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To generate pAAV-EF1a-DIO-hOPN4-mCherry, pAAV-EF1a-DIO-hM3Dq-mCherry (addgene#50460) was modified. The coding sequences of hM3Dq was replaced by the sequence coding human OPN4 isoform1 (Genbank: NM_033282) in pAAV-GFAP104-melanopsin-mCherry (addgene#122630).50 (link) Amino acid from 385 to 478 and from 400 to 478 was deleted for hOPN4-dC and hOPN4-9A-dC, respectively. For hOPN4-9A and hOPN4-9A-dC, synthesized oligo nucleotides with mutations from serine/threonine to alanine (SA/TA) were inserted into pAAV-EF1a-DIO-hOPN4-mCherry by In-Fusion HD Cloning Kit (Z9648N, Clontech Laboratories Inc.) at the position of amino acid 384, 387, 388, 391, 392, 394, 395, 397 and 398. pEF1α-hOPN4-mCherry and pEF1α-hOPN4dC-mCherry was generated by insertion of the coding sequences of hOPN4-mCherry and hOPN4dC-mCherry into pEF1α-mCherry-N1 (Clontech Laboratories Inc) by In-Fusion HD Cloning Kit (Z9648N, Clontech Laboratories Inc). pGL4.30-NFAT-Luc vector was purchased ([luc2P/NFAT-RE/Hygro], E8481, Promega Corp.).
4
Assessing NFAT Transcriptional Activity
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Jurkat cells were collected 48 h after transfection with pX458 and pGL4.30 (luc2P/NFAT-RE/Hygro, Promega, Madison, WI, USA) vectors and assayed for NFAT luciferase activity using the Dual-Luciferase Reporter Assay System (Promega) and Wallac ARVO SX 1420 Multilabel Counter (PerkinElmer, Waltham, MA, USA). Firefly luciferase activity was normalized by Renilla luciferase activity (phRL-TK vector, Promega) in each sample, and is presented with a logarithmic scale relative to the activity in mock-transfected cells.
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