The liposome-mediated transduction was conducted as described previously16 (link). Approximately 15,000 cells CHOK1 RD-GFP cells were seeded in 8-well Lab-Tek chambers (Thermo Fisher Scientific) in the presence of 1 µg/ml doxycycline. The next day, for transducing 2 wells of an 8-well, 24 µl of Opti-MEM I Reduced Serum (Thermo Fisher Scientific) medium was supplemented with 1 µl of Lipofectamine-2000 and these were mixed with 25 µl Opti-MEM I Reduced Serum medium containing the sonicated tau fibrils. As control, monomeric tau E14 was employed. The protein-liposomes mix was incubated at RT for 20 min and then 20 µl for every well were applied drop-wise to the cells. The final concentration of tau fibrils or monomer applied to the cells was 400 nM. About 16 h later, the cells were fixed and processed for analysis.
Opti mem 1 reduced serum
Manufactured by Thermo Fisher Scientific
Sourced in United States
Opti-MEM I Reduced Serum is a cell culture medium designed to support the growth and maintenance of various cell types in a serum-reduced environment. It is formulated to provide essential nutrients and growth factors while minimizing the concentration of serum components.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (9 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (4 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (3 mentions)
24 well plate, by Thermo Fisher Scientific (2 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Lab tek chamber, by Thermo Fisher Scientific (1 mentions)
Amantadine, by Merck Group (1 mentions)
40 um cell strainer, by BD (1 mentions)
Mirna oligonucleotides, by GenePharma (1 mentions)
Peg it virus precipitation solution, by BioCat (1 mentions)
Estrogen, by Merck Group (1 mentions)
Cyclic adenosine monophosphate, by Merck Group (1 mentions)
Progesterone, by Merck Group (1 mentions)
Gnrha, by Merck Group (1 mentions)
Opti mem 1, by Thermo Fisher Scientific (1 mentions)
Scrambled sirna, by Thermo Fisher Scientific (1 mentions)
Scramble sirna, by Thermo Fisher Scientific (1 mentions)
X tremegene 9 transfection reagent, by Roche (1 mentions)
16 protocols using opti mem 1 reduced serum
1
Liposome-mediated Tau Fibril Transduction in CHO Cells
2
Inducing Neuro-2A Cell Differentiation
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Neuro-2A cells and HeLa cells were respectively cultured in Dulbecco’s modified Eagle medium (DMEM, Thermo Fisher scientific) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher scientific) at 37 °C and in 5% CO2. Transiently transfection of cells with plasmid DNA was performed using Lipofectamine® 2000 Transfection Reagent (Thermo Fisher scientific) in Opti-MEM® I Reduced Serum (Thermo Fisher scientific), by following to the manufacturer’s instructions. For induction of differentiation, Neuro2A cells were transiently transfected as mentioned above. 24 h after transfection, the medium was carefully replaced with an equal volume of DMEM with 1% fetal bovine serum and supplemented with 10 μM Retinoic acid (RA) for another 48 h to induce neurite outgrowth.
3
Quantifying Internalization Ratio of Radiolabeled Compounds
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Internalization ratio was determined according to the literature [45 (link),46 (link)]. Prior to seeding cells, 24-well plates were coated with 0.1% poly-l -lysine (Sigma-Aldrich) in PBS for 20 min at room temperature. Subsequently, 105 LNCaP cells in 1 mL RPMI 1640 Medium were added in each well and incubated for 24 h at 37 °C. Then, 250 µL of the 68Ga-labeled compounds in Opti-MEM™ I Reduced Serum (ThermoFisher) were added to each well to a final concentration of 30 nM. The plates were then incubated for 45 min at 4 °C and 37 °C respectively either with or without adding PMPA (Sigma-Aldrich) to a final concentration of 500 µM. The supernatant was removed and the cells were washed several times with ice-cold PBS. Afterwards, cells were incubated twice with 50 mM glycine buffer pH 2.8 for 5 min to remove the surface-bound radioactivity. In order to determine the internalized fraction of the compounds, cells were lysed by incubation with 0.3 M NaOH for 10 min.
4
Lipofectamine 2000 Transfection Protocol
5
Bicistronic Construct for EV71 IRES Analysis
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The bicistronic construct of EV71 IRES (Fig. 7A ) was a kind gift from Professor Peter C McMinn, University of Sydney. The construct contains the 5’ untranslated region (UTR) of the EV71–26M strain and two reporter genes, Renilla luciferase (RLuc) and firefly luciferase (FLuc). The RLuc-reporter gene was positioned upstream of the EV71 5’ UTR controlled by the cytomegalovirus promoter (CMV). The firefly luciferase (FLuc) reporter gene is ligated downstream of the 5’ UTR which controls its expression [67 (link)].
1μg of plasmid DNA was introduced into RD cells using 2μL of Lipofectamine 2000 (Invitrogen) in Opti-MEM I Reduced serum (Gibco) according to manufacturer’s instructions. The cells were incubated for 12h and the media was replaced with DMEM 2% FCS growth medium. As a negative control, 0.5mg/ml of amantadine (Sigma-aldrich) was added to the media of untreated cells to inhibit the EV71 IRES activity. The cells were incubated at 37°C for another 12h before harvesting.
1μg of plasmid DNA was introduced into RD cells using 2μL of Lipofectamine 2000 (Invitrogen) in Opti-MEM I Reduced serum (Gibco) according to manufacturer’s instructions. The cells were incubated for 12h and the media was replaced with DMEM 2% FCS growth medium. As a negative control, 0.5mg/ml of amantadine (Sigma-aldrich) was added to the media of untreated cells to inhibit the EV71 IRES activity. The cells were incubated at 37°C for another 12h before harvesting.
6
Lipofectamine-Mediated Transfection Workflow
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One day prior transfection, cells were passed through a 40um cell strainer (Falcon; Cat. No 352340) and counted with Bio Rad TC10. In each well (uncoated 6-well plates, Thermo Scientific Nunc; Cat. No. 2020–10) 300,000 cells were seeded and incubated for another 24 hours. On the day of transfection DNA was diluted in 250ul Opti-MEM I Reduced Serum (Gibco, Life Technologies Cat no. 31985–962) and mixed with a 244ul Opti-MEM I/6ul Lipofectamin 2000 Transfection Reagent (Thermo Fischer; Cat. no. 11668019). After a 20 minutes’ incubation step at room temperature, the transfection mix was added drop wise to the wells. The cells were incubated for another 72 hours before being measured by flow cytometry.
7
Transfection Protocol for Cell Lines
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All transfections were performed using Lipofectamine 2000 Transfection Reagent (Life Technologies) in uncoated 24-well plates (Thermo Scientific Nunc). One day before transfection cells were seeded into 500 μl medium per well at a cell densitiy of 7.5 × 104 for HEK293 and 6.5 × 104 for HeLa and HuH-7 per well. Medium was replaced with medium supplemented with Doxycycline hyclate (Fluka, Cat # 44577) at a final concentration of 1 μg/ml shortly before transfection if required. Transfection was performed at 80 - 90% cell confluence and low passage number. The plasmids were mixed according to Supplementary Tables 1-9 and diluted with 50 μl Opti-MEM I Reduced Serum (Gibco, Life technologies Cat # 31985-962) per sample. If needed, miRNA mimics miR-21, miR-141, miR-146 and miR-Ctrl were added to the plasmid mix. All mimics were purchased from GE Healthcare. Exact microRNA mimic specifications can be found in Supplementary Table 17 . Lipofectamine 2000 was used in amounts of 1.5 μl (Fig. 2 , 3b , Supplementary Figs. 1, 2, 3 ,) or 1.8 μl (Figs. 3c , 4 -8 , Supplementary Fig. 6 ) per sample and was mixed with 50 μl Opti-MEM. After 3 minutes incubation at room temperature, the diluted Lipofectamine was mixed with the diluted DNA sample. The mixture was incubated for 15 min at room temperature and added to the cells.
8
Osteoblast Differentiation: miR-539-3p Modulation
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MCOs were seeded 1 day prior to the transfection in cell culture plates in osteoblast growth medium. Cells at 60-70% confluence were transfected with oligo miRNAs like miC (negative control), miR-539-3p (mimic) and anti-miR-539-3p (inhibitor) and siRNAs purchased from Thermo Fisher Scientific, USA by using lipofectamine 2000 (Thermo Fisher Scientific, USA) transfection reagent in Opti-MEM®I reduced serum (Gibco, Life technologies, USA) medium as per manufacturer’s protocol. Sequences are given in Supplementary Table 1
9
Cell Culture and Transfection Protocols
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Neuro-2A cells, SH-SY5Y cells and HeLa cells were cultured in Dulbecco’s modified Eagle medium (Gibco, United States) supplemented with 10% fetal bovine serum (Gibco, United States) at 37°C and 5% CO2. Cells were transiently transfected using Lipofectamine 2000 (Invitrogen, United States) in Opti-MEM I Reduced Serum (Gibco, United States), according to the manufacturer’s instructions. After transfection for 24 h or 36 h, cells were harvested for RNA/protein extraction or immunofluorescent labeling.
10
Overexpressing miR-136 in Cells
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Synthesized miR-136 mimic (GenePharma, Suzhou, P.R. China) or mimic negative control (NC) were transfected into cells with Lipofectamine® 2000 (Invitrogen, Carlsbad, CA, USA), which was mixed in Opti-MEM® I reduced serum (Gibco, Grand Island, NY, USA) according to the manufacturer’s protocol. qPCR was performed to measure the expression of miR-136 in cells following transfection. All miRNA oligonucleotides were purchased from GenePharma (Shanghai, P.R. China).
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