pAAV-VGF-GFP and pZsGreen1-N1 (a positive control; Clontech Laboratories, CA, USA) plasmids were transfected into undifferentiated SH-SY5Y cells using FuGENE HD (Promega, Wisconsin, USA) according to the manufacturer's protocol. Briefly, prior to transfection, cells at approximately 80% confluence were transferred to OptiMeM1® reduced serum media containing no antibiotics (Promega, Wisconsin, USA). Plasmid DNA was diluted in OptiMEM1® to give a concentration of 20 μg/ml. The FuGENE® HD transfection reagent was added to achieve a 3:1, reagent: DNA ratio and 10 μl of the reagent:DNA mixture was added to each well equating to 50 ng of plasmid DNA per well. The cells were incubated for 72 h prior to assessment of eGFP fluorescence and VGF mRNA expression. Cells transfected in parallel were further incubated with RA (10 μM) to induce differentiation prior to eGFP assessment.
Opti mem 1
Manufactured by Promega
Sourced in United States
Opti-MEM I is a cell culture medium designed to support the growth and maintenance of a variety of cell lines. It is a serum-reduced medium that provides essential nutrients and growth factors to cells. Opti-MEM I is commonly used in transfection experiments and other applications that require minimal serum conditions.
Automatically generated - may contain errors
Lab products found in correlation
Vivazine substrate, by Promega (2 mentions)
Transfection carrier dna, by Promega (2 mentions)
Opti mem 1, by Thermo Fisher Scientific (2 mentions)
Pzsgreen1 n1, by Takara Bio (1 mentions)
Fugene hd, by Promega (1 mentions)
Trypsin edta, by Innovative Cell Technologies (1 mentions)
Oligofectamine, by Promega (1 mentions)
C fos, by Santa Cruz Biotechnology (1 mentions)
Prl tk renilla luciferase vector, by Promega (1 mentions)
Q5 hot start high fidelity 2x master mix, by New England Biolabs (1 mentions)
Trans it 293 reagent, by Promega (1 mentions)
Pgl3 basic firefly luciferase reporter vector, by Promega (1 mentions)
Dual glo luciferase assay system, by Promega (1 mentions)
6 protocols using opti mem 1
1
Transfection and Differentiation of SH-SY5Y Cells
2
CD32B-SHIP-1 Binding Assay
3
Astrocyte Transfection of Signaling Receptors
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As described previously (24 (link)), the cultured astrocytes were incubated in DMEM without serum for half day before transfection. A transfection solution containing 2 μl oligofectamine (Promega, Madison, WI, USA), 40 μl Opti-MEMI, and 2.5 μl siRNA (666 ng) was added to the culture in every well for 8 h. In the siRNA-negative control cultures, transfection solution without siRNA was added. Thereafter, DMEM with three times serum was added to the cultures. The siRNA duplex chains of leptin receptor (LepR), 5-HT2B receptor (5-HT2BR) and c-fos were purchased from Santa Cruz Biotechnology (CA, USA).
4
KRAS(G12D):CRAF-RBD NanoBiT Interaction Assay
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For the KRAS(G12D):CRAF-RBD NanoBiT interaction assay, cytomegalovirus-based expression constructs were made encoding fusions of LgBiT to KRAS 4B (UniProt P01116-2) with the G12D mutation and SmBiT to residues 51−133 of CRAF (UniProt P04049-1, CRAF(RBD)). HEK293 cells (~4 × 106) were transiently transfected in T75 flasks with plasmids encoding a LgBiT-KRAS(G12D) and SmBiT-CRAF(RBD). Plasmids were transfected at 500 ng per construct per flask together with 9 µg of Transfection Carrier DNA (Promega) at a 3:1 lipid:DNA ratio using FuGENE HD (10 ml total volume). Following expression for 24 h, cells were plated at 20,000 cells per well in Opti-MEM I (Thermo) containing 4% FBS and allowed to attach overnight. Serial dilutions of MRTX-EX185 were made in Opti-MEM I containing 4% FBS and 1× Vivazine substrate (Promega N2581) to generate 1× solutions containing varying concentrations of MRTX-EX185. Existing medium was removed by plate inversion and blotting, and 1× solutions were added to respective wells. Luminescence was measured every 5 min in a GloMax Discover luminometer at 37 °C for 16 h using a 1 s integration time.
5
Regulation of Tgfbr2 promoter by ZBTB18
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The mouse Tgfbr2 promoter (702 bp, from −650 to +52 relative to the transcription start site) was amplified from genomic DNA of E0771GFP cells by PCR using Q5 Hot Start High-Fidelity 2X Master Mix (NEB). The Tgfbr2 promoter fragment was cloned into the Kpn I and Hind III restriction sites of the pGL3-Basic firefly luciferase reporter vector (Promega). A mutant Tgfbr2 promoter where putative ZBTB18 binding sites (5′-[AC]ACATCTG[GT][AC]-3′) are substituted with two Sma I binding sites (5′-CCCGGGCCCGGG-3′) was generated by cloning a gBlock into the Kpn I and Hind III restriction sites of the pGL3-Basic firefly luciferase reporter vector. 293FT cells were plated in 96-well plates at 104 cells per well. After 24 hours, cells were cotransfected with 75 ng of luciferase reporter plasmid (pGL3-Basic, pGL3-Tgfbr2 promoter, or pGL3-mutant Tgfbr2 promoter); 75 ng of expression plasmid ZBTB18OE, ZBTB18-Nucl, ZBTB18-Cyto, or empty expression vector (EV); and 50 ng of pRL-TK Renilla luciferase vector (to normalize for transfection efficiency; Promega) using TransIT-293 reagent in Opti-MEM I. After 24 hours, luciferase activity was measured using the Dual-Glo Luciferase Assay System (Promega). Firefly luciferase values were normalized to Renilla luciferase values and protein content and reported relative to the EV condition.
6
KRAS(G12D):CRAF RBD NanoBiT Assay
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