To determine the effects of exogenous host proteases expression on coronavirus replication, plasmid DNA of human CTSL/TMPRSS2/Furin was transfected into cells before viral infection. 1 μg plasmid DNA was pre-incubated with 0.2 μL P3000 reagent (Invitrogen) in 50 μL Opti-MEM Reduced Serum Media (Invitrogen) at room temperature for 5 min, followed by mixed with another 50 μL Opti-MEM Reduced Serum Media consisting of 1 μL lipofectamine3.0 transfection reagent (Invitrogen). After 0.5 h of incubation, the transfection mixture was added into cells at 70% confluence for 24 h at 37 °C. Then 1 mL of maintenance medium (2% FBS) containing HCoV-229E or HCoV-OC43 (MOI = 0.1) and test compounds was used to treat cells for 24 h at 35 °C. Total cellular proteins were extracted for Western blot assay after infection.
Opti mem reduced serum media
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, China
Opti-MEM reduced serum media is a cell culture medium designed to support the growth and maintenance of a variety of mammalian cell types. It contains a reduced concentration of serum components compared to traditional cell culture media, which may be beneficial for specific applications.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (38 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (20 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (18 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (18 mentions)
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (16 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (7 mentions)
Blasticidin, by Thermo Fisher Scientific (6 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (6 mentions)
White 384 well plate, by PerkinElmer (6 mentions)
Envision multilabel plate reader, by PerkinElmer (6 mentions)
Britelite plus, by PerkinElmer (6 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (5 mentions)
Rnaimax, by Thermo Fisher Scientific (5 mentions)
Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (5 mentions)
Dharmafect 1, by Horizon Discovery (4 mentions)
Penicillin streptomycin, by Merck Group (4 mentions)
Puromycin, by Thermo Fisher Scientific (4 mentions)
Hek293t cell, by American Type Culture Collection (4 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (4 mentions)
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (4 mentions)
241 protocols using opti mem reduced serum media
1
Exogenous Protease Regulation of Coronavirus Replication
2
HeLa-Kyoto and RPE-1 Cell Culture and Transfection
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Human HeLa-Kyoto (RRID:CVCL_1922 , gift of Ryoma Ohi, University of Michigan) and RPE-1 cell lines (ATCC #CRL-4000, RRID:CVCL_4388 ) were grown in Minimum Essential Media α (Gibco #12561-056) supplemented with 10% fetal bovine serum (FBS; Gibco #16000-044) at 37°C with 5% CO2. Cell lines were validated by short tandem repeat (STR) DNA typing using the Promega GenePrint 10 System according to the manufacturer’s instructions (Promega #B9510). Cells were cryopreserved in Recovery Cell Culture Freezing Medium (Gibco #12648-010). HeLa-Kyoto and RPE-1 acceptor cell lines for recombination (both gifts from Ryoma Ohi, University of Michigan) were maintained in media supplemented with 10 μg/ml blasticidin (Thermo Fisher Scientific #R21001).
Transfection siRNA transfection was performed using Lipofectamine RNAiMax Transfection Reagent (Thermo Fisher Scientific #13778150) in Opti-MEM Reduced Serum Media (Gibco #31985-062). KIF22 was targeted for siRNA-mediated depletion using a Silencer Validated siRNA (Ambion #AM51331, sense sequenceGCUGCUCUCUAGAGAUUGCTT ). Control cells were transfected with Silencer Negative Control siRNA #2 (Ambion #AM4613). DNA transfections were performed using Lipofectamine LTX (Thermo Fisher Scientific #15338100) in Opti-MEM Reduced Serum Media (Gibco #31985-062).
Transfection siRNA transfection was performed using Lipofectamine RNAiMax Transfection Reagent (Thermo Fisher Scientific #13778150) in Opti-MEM Reduced Serum Media (Gibco #31985-062). KIF22 was targeted for siRNA-mediated depletion using a Silencer Validated siRNA (Ambion #AM51331, sense sequence
3
siRNA Knockdown in Fibroblasts
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Lipofectamine RNAiMAX (Thermo Fisher Scientific, 13778150) transfection was performed following the manufacturer’s protocols. In brief, 500 µl of Opti-MEM Reduced Serum Media (Thermo Fisher Scientific, 31985070) containing either Dharmacon’s siRNA targeting H1.0 (40 nM, Horizon Discovery, M-060325-01), H1.1 (80 nM, Horizon Discovery, M-049956-00), H1.2 (80 nM, Horizon Discovery, M-045246-00), H1.3 (80 nM, Horizon Discovery, M-051171-00), H1.4 (80 nM, Horizon Discovery, M-042536-01), H1.5 (80 nM, Horizon Discovery, M-049995-00) and Thbs4 (40 nM, Horizon Discovery, M-044016-01) or the appropriate concentration of siRNA scramble control (Horizon Discovery, D-001206-14) were mixed with 500 µl of Opti-MEM Reduced Serum Media containing 20 µl of Lipofectamine RNAiMAX (Thermo Fisher Scientific, 13778075). For the human skin fibroblast experiment, transfection was performed using Dharmacon’s siRNA targeting human H1.0 (40 nM, Horizon Discovery, M-017209-01). After incubating the reagents for 10 min at 37 °C, the solution was added to the cells and slightly agitated to mix. After 24 h of incubation at 37 °C, the siRNA reagent solution was removed and replaced with appropriate media according to the downstream experiment. siRNA sequences are listed in Supplementary Table 2 .
4
Directed Differentiation of MEFs into iHeps
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To generate R-iHeps, mouse embryonic fibroblasts (MEFs) were cultured in Dulbecco's Modified Eagle's Medium (Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum, 3.14 uM β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA), and 1% penicillin/streptomycin (Life Technologies) at 37°C in a CO2 incubator. Lipofectamine 2000 (Life Technologies) was used for mRNA transfection. On day 0 and 3 , 1.5 ug of Foxa3 and HNF4α mRNA each and 3 ul of Lipofectamine 2000 were diluted in a mixture of 125 ul of Opti-MEM reduced serum media (Life Technologies) in separate tubes. They were then mixed together into one tube and were incubated at room temperature for 5 minutes. In a culture dish, 250 ul of the incubated mixture was added in 1ml of cell growth media and was incubated at 37°C for 4 hours. After 24 hours, the medium was changed with DMEM/F-12 (Life Technologies) supplemented with 10% fetal bovine serum (Life Technologies), 10mM Nicotinamide (Sigma-Aldrich), 0.1 uM dexamethasone (Sigma-Aldrich), 1% Insulin-Transferrin-Selenium-X Supplement (Life Technologies), 1% penicillin/streptomycin (Life Technologies), 20 ng/ml hepatocyte growth factor (Peprotech, Rocky Hill, NJ, USA), and 20 ng/ml epidermal growth factor (Peprotech). The medium was changed every two days.
5
Recombinant SLC1A5 Protein Expression
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The gene encoding SLC1A5 was cloned into pcDNA3.1+ (Life Technologies) where it was fused to a C-terminal His10-Flag affinity double tag separated by TEV protease cleavage site. The protein was expressed in HEK293S GnTI- cells (ATCC CRL-3022, RRID: CVCL_A785 ) grown in Expi293 Expression Media (Life Technologies) to densities 2.5 × 106 cells ml−1. Cells were transiently transfected in Opti-MEM reduced serum media (Life Technologies) using ExpiFectamine Transfection Kit (Life Technologies). Cells were collected at 48 hr after transfection. Although commercially available, MAb cKM4012 (Creative Biolabs Inc) was cloned, expressed and purified in-house to meet reagent quantity requirements (Suzuki et al., 2017 (link); Shiraishi et al., 2012 ).
6
Knockdown of PDGF-B and PRDX5 by shRNA
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The gene sequence of PDGF-B and PRDX5 was obtained from Genbank (Gene ID: 5155 and 25824). Two sequence-specific shRNAs were designed based on the rules as described elsewhere. The shRNA expressing plasmids were constructed by GenePharma Corporation (Shanghai, China) using pGPU6/GFP/Neo vector. The sequences of the shRNAs targeting PDGF-B and PRDX5 were 5′- GCGGAAGAAGCCAATCTTTAA-3′ and 5′-GCCTGGCACCCAATATCATCT-3′, respectively. An unrelated shRNA sequence with no homology to any human gene was used as a negative control. Transfection was performed using OPTI-MEM® reduced serum media (Life Technologies, Carlsbad, CA, USA) according to the manufacturers’ instructions.
7
Arrb1 Knockdown in N-38 Neurons
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To knockdown the expression of Arrb1 in N-38 neurons, Arrb1 siRNA (cat. # SI02699116) and scrambled siRNA (cat. # 1027310) were purchased from Qiagen (Valencia, CA). Transient transfections were performed using Lipofectamine RNAiMax (Life Technologies, Grand Island, NY) with 50 nM scrambled or Arrb1 siRNA in OptiMEM reduced serum media (Life Technologies). Forty-eight hours after transfections, cells were steroid-starved for 20 h in charcoal-stripped media (phenol red-free DMEM supplemented with 5% charcoal-stripped/dextran-treated FBS (Gemini Bio-Products, West Sacramento, CA), 4.5 mg/ml glucose, 1% penicillin/streptomycin, 0.15% sodium bicarbonate) prior to biotinylation or internalization experiments.
8
Plasmid and siRNA Transfection Protocol
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Plasmid or siRNA transfection using lipofectamine (Life Technologies - Invitrogen) and OPTI-MEM reduced serum media (Life Technologies - Invitrogen) was performed as described previously [46 (link)]. The cells were transfected after plating for 24 h. After a further incubation of 24 h, the cells were seeded onto a 96-well plate for growth curve analysis. The sequences of ARID1A siRNAs were synthesized as previously reported [22 (link)]. The siRNAs were listed in Supplementary Table 1 .
9
EGFR Activation Protocol
10
Adipocyte Knockdown of Key Signaling Proteins
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IKKβ, JNK1, PKCθ, and control siRNA (10 μmol/l) were added to OPTI-MEM-reduced serum media (Life Technologies) with Lipofectamine RNAiMAX (Invitrogen Corp.). Adipocytes in six-well plates were transfected with siRNA in transfection medium for 6 h. The transfection medium was then replaced with a culture medium containing 10% FBS and incubated for 48 h.
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