HeLa cells grown on glass coverslips (Matsunami) in a 6-well plate were transfected with Lyn11-Halotag using Lipofectamine 2000 (Invitrogen). Afters 4 h, the culture medium was replaced with DMEM and the cells were incubated at 37 °C for 24 h. The culture medium was exchanged with Opti-MEM (Gibco), and the cells were incubated at 37 °C. After 2 h, the cells were washed once with Opti-MEM and incubated with 0.03 nM of Halotag TMR ligand (Promega) in Opti-MEM for 30 min in a CO2 incubator. The cells were then washed three times with Opti-MEM and single-molecule imaging was performed using a TIRF microscope. Single particle detection and estimation of diffusion constants were done using ICY and PNN algorithm, respectively.
Opti mem
Manufactured by Promega
Sourced in United States
Opti-MEM is a cell culture media used to support the growth and maintenance of various cell lines. It is designed to provide a serum-reduced or serum-free environment for cell transfection and other applications. Opti-MEM is a balanced salt solution that contains essential nutrients, vitamins, and other components necessary for cell survival and proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Opti mem, by Thermo Fisher Scientific (23 mentions)
Fugene hd, by Promega (12 mentions)
Fugene, by Promega (11 mentions)
Passive lysis buffer (plb), by Promega (5 mentions)
Lipofectamine 2000, by Promega (4 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (4 mentions)
Dual luciferase reporter assay system, by Promega (3 mentions)
Fugene 6, by Promega (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Pluronic acid, by Thermo Fisher Scientific (2 mentions)
Fura 2 am, by Thermo Fisher Scientific (2 mentions)
Hek293t cell, by American Type Culture Collection (2 mentions)
Hek293t cells, by Promega (2 mentions)
Dual glo luciferase assay system, by Promega (2 mentions)
Fugene 6 transfection reagent, by Promega (2 mentions)
Pgl4.74 hrluc tk, by Promega (2 mentions)
Luciferase assay system, by Promega (2 mentions)
Viafect transfection reagent, by Promega (2 mentions)
Pgl4.23firefly luciferase reporter vectors, by Promega (2 mentions)
54 protocols using opti mem
1
Single-molecule Imaging of Lyn-Halotag
2
Overexpression of CD44 Isoform 12 in MB-231 Cells
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MB-231 cells were transformed with the plasmid pLX304 expressing CD44 isoform 12 under the CMV promoter and a C-terminal V5 tag as previously described [33 (link),42 (link)]. In brief, 5 × 104 cells were plated in each well of a 12 well plate and allowed to attach overnight in growth medium. The medium was then replaced with OptiMEM (Life Sciences, Carlsbad, CA) for 24 h. To transform cells, the medium was changed to 500 µl OptiMEM medium supplemented with 20 µl of medium containing 5 µl of Fugene (Promega, Madison, WI), 2 µg of pLX-304-CD44 isoform 12 and 98 µl of OptiMEM. Cells were allowed to grow overnight and maintained in 10 µg/ml selection drug Blasticidin S (Enzo Life Sciences, NY).
3
HEK293T Transfection and NLRP1 Activation
4
Lentivirus Production and Purification
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Lentiviruses were developed by transfecting HEK293T LentiX cells with lentiviral packaging (psPAX2; Addgene #12260, gift from Didier Trono, Swiss Federal Institute of Technology Lausanne, Switzerland) and envelope (pDM2.G; Addgene #12259, gift from Didier Trono) plasmids alongside NMIIB constructs. Briefly, psPAX2 (16.876 μg), pMD2.G (2.767 μg), and NMIIB/GFP control constructs (6.548 μg) were added to 500 μl of Opti-MEM (Life Technologies #31985070). Then 78.57 μl of FuGENE (Promega #E2311) was added to 421.4 μl of Opti-MEM in a separate tube before mixing with DNA containing Opti-MEM followed by brief vortexing. The mixture was then allowed to sit at room temperature for 10 min before being added dropwise to HEK293T LentiX cells at approximately 70% confluency in media without penicillin or streptomycin. Cells were incubated overnight before media was changed and then allowed to incubate for 2 d after which the media was collected and filtered through a 0.45 μm syringe filter to remove cells and then centrifuged at 16,600 rpm at 4°C for 2 h. All but ∼500 μl of the supernatant was aspirated, and the viral pellet was left to incubate overnight at 4°C before resuspending the pellet and snap freezing in 100 μl aliquots.
5
Methylation Impacts on TERT Promoter Activity
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TERT promoter regions were cloned into a pNL1.1 vector (Promega), transformed in TOP10 chemically competent cells (Lifetech), and verified by Sanger sequencing. To generate methylated versus unmethylated plasmids, 1 μg of plasmid was treated with SssI (NEB) or mock as per manufacturer’s instructions. To ensure complete methylation, plasmid DNA was treated with SssI twice. Cells in culture were seeded into 96 well plates and transfected in suspension using X-tremeGENE HP DNA Transfection Reagent (Roche). Transfection complexes were prepared such that each reaction contained 11 μl Opti-MEM, 59 ng empty pCR2.1-TOPO vector, 5 ng pGL4.53(luc2/PGK) vector (Promega), 1 ng pNL plasmid, and 0.12 μl HP transfection reagent. After 2 days, transfected cells were lysed in 50 ul passive lysis buffer (Promega), transferred to black 96 well plates, and measured for reporter activity by mixing 50 μl of ONE-Glo EX Luciferase Reagent and NanoDLR Stop & Glo Reagent sequentially as described in the Nano-Glo Dual Luciferase Assay System manual (Promega).
6
Establishing Stable Transfection of EGFP
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Cells (2 x 105) were cultured in 6-well plates in 10% FBS-DMEM for 2 days. The medium was replaced with 800 μL of OPTI-MEM (Invitrogen), and then 200 μL of OPTI-MEM containing 1 μg of pEGFP-C1 vector (BD Biosciences), 4 μL of Lipofectamine Reagent (Invitrogen) and 6 μL of PLUS Reagent (Invitrogen) were added to the cells. After a 3 h incubation, 1 mL of 20% FBS-OPTI-MEM was added to each well and the cells were cultured for 24 h. Stably transfected cells were selected with 400 μg/mL of G418 (Promega).
7
Evaluating Measles Virus Fusion Activity
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Cells (5×105) were seeded onto 6-well plates (Costar Corp) the day before the experiment. Two hours before transfection, the culture medium was removed and replaced with fresh medium without antibiotics. Then, equal amounts (1 μg) of pCG plasmids encoding MeV-F and the indicated MeV-H mutants or pCG-MeV-H alone (2 μg) were diluted in 100 μL of OptiMEM (ThermoFisher) onto a 96-well plate. Another 100 μL of OptiMEM containing 6 μL of Fugene HD (Promega) was then added to each well, and the mixture was incubated for 20 min. The solution was then added to the cells.
Fusion activity was evaluated 24 h later after Hema-Quik staining (Fisher Scientific). The size of the syncytia was quantitated using NIS-Elements microscope imaging software (Nikon). Alternatively, the number of nuclei in randomly chosen syncytia was counted.
To harvest protein lysates, cells were washed with phosphate-buffered saline (PBS) before they were lysed on ice for 15 min with 0.5 mL RIPA buffer (Abcam) containing Halt protease inhibitor cocktail (ThermoFisher). The supernatant containing the cleared lysate was collected after centrifugation at 13,000 rpm at 4°C for 15 min; aliquots were kept at −20°C if not used immediately.
Fusion activity was evaluated 24 h later after Hema-Quik staining (Fisher Scientific). The size of the syncytia was quantitated using NIS-Elements microscope imaging software (Nikon). Alternatively, the number of nuclei in randomly chosen syncytia was counted.
To harvest protein lysates, cells were washed with phosphate-buffered saline (PBS) before they were lysed on ice for 15 min with 0.5 mL RIPA buffer (Abcam) containing Halt protease inhibitor cocktail (ThermoFisher). The supernatant containing the cleared lysate was collected after centrifugation at 13,000 rpm at 4°C for 15 min; aliquots were kept at −20°C if not used immediately.
8
TRPV2 Calcium Imaging in HEK293T Cells
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Wild-type rabbit TRPV2 and the truncated TRPV2 construct were expressed in HEK293T cells (ATCC). HEK293T cells were plated on glass coverslips and transiently transfected with a mixture of DNA, Opti-MEM and FuGENE (Promega) according to the manufacturer's manual. Cells were seeded at 60 × 103 per well in the presence of 10 μM ruthenium red and incubated for 48 h at 37 °C. The transfected cells were washed in Hank's buffer and incubated with 2 μM Fura-2 AM in the presence of pluronic acid (Invitrogen) for 30–60 min. Imaging was performed as previously described56 (link). In brief, frames were recorded every second at 340 nm and 380 nm, and the data were analyzed with Nikon Elements software.
9
Lentiviral Transduction of Target Cells
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With a second-generation system based on pCMVdR8.1 and pVSV-G, lentiviral particles were generated to transduce cells. For virus production, 1 × 106 HEK293T were plated in a well of a 6-well plate. Vectors of 1200 ng pLenti-plasmid, 1000 ng pCMVdR8.1, and 400 ng pVSV-G were mixed into 100 μL OptiMEM with 7.8 μL Fugene (catalogue number E3211; Promega, Madison, WI). After 5 minutes of incubation, the mixture was added into the medium of HEK293T. The next morning the medium was removed, and virus production medium: DMEM with 10% fetal calf serum and Dulbecco modified Eagle medium: F12 (no serum) in the ratio of 1:3 was added. After a 2-day incubation the virus-containing supernatant was filtered through a polyvinylidene difluoride membrane–based 0.45 μm filter (GE Healthcare, Hatfield, UK) and harvested as viral particles. After seeding in 6-well plate at least for 4 hours, the target cells were cultured with 500 μL viral particles and 1 mL medium supplemented with 8 μg/mL polybrene. After overnight incubation, the virus-containing medium was replaced with antibiotics-containing medium (2.5 μg/mL puromycin, 4 μg/mL blasticidin).
10
Investigating PGRMC2 Role in P4 Withdrawal
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