The protocol for the association of FlAsH-EDT2 with β-tubulin-TC in cell was adapted from the previous study (Hoffmann et al., 2010 (link)) so as to maximize the labeling fraction while maintaining cell viability. The engineered U2OS cells expressing β-tubulin-TC were grown to 80~90% confluency in a 30 mm cell culture dish, and then were gently washed with Opti-MEM (Thermo Fisher) twice, and then stained in 2 ml Opti-MEM with 1 μM FlAsH-EDT2 (Thermo Fisher) for 2 hr. To reduce the non-specific binding of FlAsH, the stained cells were subsequently incubated in Opti-MEM containing 250 μM 1,2-Ethanedithiol (EDT, Alfa Aesar) for 10 min, followed by a gentle wash with Opti-MEM. The cells were incubated in DMEM with 10% FBS for 6~10 hr before imaging, because they were found to be interphase-arrested for the first ~5 hr after the incubation with 250 μM EDT. Every buffers and media above were pre-warmed at 37°C before use. All incubation steps were performed at 37°C in a humidified atmosphere with 5% CO2.
Opti mem
Manufactured by Thermo Fisher Scientific
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Opti-MEM is a cell culture medium designed to support the growth and maintenance of a variety of cell lines. It is a serum-reduced formulation that helps to reduce the amount of serum required for cell culture, while still providing the necessary nutrients and growth factors for cell proliferation.
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Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (3196 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1726 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1184 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (716 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (542 mentions)
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (416 mentions)
Rnaimax, by Thermo Fisher Scientific (408 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (392 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (294 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (250 mentions)
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (236 mentions)
Glutamax, by Thermo Fisher Scientific (193 mentions)
Lipofectamine, by Thermo Fisher Scientific (191 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (170 mentions)
Penicillin, by Thermo Fisher Scientific (165 mentions)
Streptomycin, by Thermo Fisher Scientific (162 mentions)
Polybrene, by Merck Group (161 mentions)
Fugene hd, by Promega (147 mentions)
Pspax2, by Addgene (126 mentions)
Lipofectamine ltx, by Thermo Fisher Scientific (118 mentions)
8 992 protocols using opti mem
1
Optimized Labeling of β-Tubulin in Cells
2
miRNA-155 Transfection in RAW264.7 Cells
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RAW264.7 cells were seeded at 1.2 x 106 cells per well in 6-well tissue culture plates 1 day prior to transfection. Biotinylated pre-miR155 oligonucleotides (Biotin-UAAUUGUGAUAGGGGUUUUGGCCUCUGACUGACUCCUACCUGUUA ) and TNFα ARE (Biotin-UUAUUAUUUAUUAUUUAUUUAUUAUUUAUUUAUUU ) were obtained from Invitrogen Life Technologies (ThermoFisher Scientific, Nepean, ON). RNA oligonucleotide was prepared in Opti-MEM (ThermoFisher Scientific, Nepean, ON) at a concentration of 0.86 μM. Lipofectamine-2000 (ThermoFisher Scientific, Nepean, ON) was diluted in Opti-MEM at 1:25 volume/volume (v/v) ratio, and was mixed with RNA oligonucleotide-Opti-MEM solution at 1:1 (v/v) ratio and incubated at room temperature for 20 minutes to allow the formation of lipofectamine-oligonucleotide complexes. After incubation, the lipofectamine-oligonucleotide solution was further diluted with 9% RPMI at 1:1.25 (v/v) ratio and 675 μl of the solution were added to each wells with cells and incubated for 6 hours in 37°C, 5% CO2 chamber. After 6 hours, the solution in the well was replaced with 1 ml of 9% FBS/RPMI to allow cells to recover overnight.
3
CRISPR-Cas9 Mediated Ager Gene Editing
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crRNA was designed to target exon 2 of the Mus musculus Ager gene (target sequence, 5ʹ-GATTGGAGAGCCACTTGTGC-3ʹ). To prepare Cas9 gRNAs, equimolar amounts of Alt-R™ crRNA and Alt-R tracrRNA (Integrated DNA Technologies, US) were mixed in IDT Duplex Buffer (30 mM HEPES, pH 7.5, 100 mM potassium acetate; Integrated DNA Technologies), heated to 95 °C, and then slowly cooled to room temperature. To generate the RNP complex, a mixture of gRNAs, diluted Cas9 enzyme, and Opti-MEM® (Thermo Fisher Scientific) was incubated for 5 min at room temperature. Lipofection was then performed in 96-well plates. Opti-MEM® containing Lipofectamine® RNAiMAX (Thermo Fisher Scientific) was combined with an equal volume of Opti-MEM containing RNP and incubated for 20 min at room temperature. After lipoplex formation, 3 × 105 mProx cells resuspended in 1 mL of DMEM + 10% FBS were added to the transfection complex. Transfection plates were incubated at 37 °C under 5% CO2/95% air. Mutation was confirmed using a Guide-it™ Genotype Confirmation Kit (TAKARA, Japan) and Sanger sequences. Fourteen bases of the target gene were deleted and the terminal codon was inserted (5ʹ-GCCACTTGTGCTAAGCTGTAA -3ʹ).
4
Efficient HEK293A Cell Transfection
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HEK293A cells (Thermo Fisher Scientific) were maintained in Dulbecco’s Modified Eagle Medium (DMEM, Nissui Pharmaceutical) supplemented with 5% fetal bovine serum (GIBCO, Thermo Fisher Scientific) and penicillin-streptomycin-glutamine (complete DMEM) at 37°C in a humidified incubator containing 5% CO2. Transfection was performed by using polyethylenimine (PEI) solution (Polyethylenimine “Max”, Polysciences). Typically, HEK293A cells were seeded in a 6-well culture plate at cell density of 2–3 × 105 cells/mL in 2 mL of the complete DMEM and cultured for one day A transfection solution was prepared by combining plasmid solution diluted in 100 μL of Opti-MEM (GIBCO, Thermo Fisher Scientific) and 100 μL of Opti-MEM containing 5 μL of 1 mg/mL PEI (Opti-MEM-PEI). The transfected cells were further incubated for 24 hours before being subjected to an assay.
5
Biosensor Cell Transduction Assay
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Biosensor cells were plated at 25,000 cells per well in 96-well plates. After 18 hours, cells were transduced with mouse or human homogenates as previously described [13 (link)]. Tissue from the mouse aging study and human samples were used at stock concentrations prepared as described above, while samples from the spreading paradigm were diluted 1:5 in TBS. Tissue samples were added to Opti-MEM (Thermo Fisher Scientific) and incubated for 5 minutes (5 μL of mouse tissue lysate with 5 μL of Opti-MEM, or 3.3 μL human with 6.7 μL of Opti-MEM per well). Lipofectamine was incubated with Opti-MEM (1.25 uL Lipofectamine with 8.75 μL Opti-MEM per well) for five minutes. Lipofectamine complexes were then mixed with samples and incubated for 20 minutes prior to addition to biosensor cells.
Mouse tissue samples were assayed in triplicate, and human tissue samples were added in sextuplicate. Cells were kept at 37 °C in a humidified incubator for 24 hours for experiments with cell lysate or transgenic mouse brain homogenate, and for 48 hours for all human tissue experiments described here. Cells were subsequently collected and prepared for flow cytometry analysis.
Mouse tissue samples were assayed in triplicate, and human tissue samples were added in sextuplicate. Cells were kept at 37 °C in a humidified incubator for 24 hours for experiments with cell lysate or transgenic mouse brain homogenate, and for 48 hours for all human tissue experiments described here. Cells were subsequently collected and prepared for flow cytometry analysis.
6
Transfection and selection of L6 cells
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Prior to transfection, low passage WT-L6 cells were cultured in a T75 flask to 90% confluency. 2 transfection mixtures were prepared. An integrase/DNA/OptiMEM mix was first created by adding FoxO1 Biosensor and SB100X plasmids at a 10:1 ratio respectively to OptiMEM (ThermoFisher; 31985062) for a total volume of 150 μL. The first mixture was then combined with the second tube containing 3.6 μL of Lipofectamine 2000 (Invitrogen; 11668019) and 146.4 μL of OptiMEM. The mixture was then vortexed for 2 s, spun down on tabletop centrifuge and incubated for 15 min at room temperature. On the other hand, L6 cells were trypsinized at room temperature for 15 min in order for the cells to detach from the bottom. The cells were then neutralized by adding AMEM media containing 10% FBS and antibiotics. 900 μL of cell and media mixture was seeded into 1-3 wells of a 6-well plate per plasmid. 100 μL of DNA and lipofectamine mix was then added in each well. Plate was gently shook to mix followed by incubation at 37 °C for 24 h. On day 2 of transfection, cells were split into a T25 flask and selected for successful transfection with 2 μg/μL of puromycin (Thermofisher Scientific; A113803) the next day. After 24 h, AMEM media with 10% FBS was changed to the flask to maintain cell proliferation. Cells were subjected to a second time of puromycin selection 7 days later.
7
Lamin A/C Silencing in mMSCs
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Lamin A/C silencing siRNA (Thermo Scientific, cat. no. D-001050-01-05) with transfection indicator was used to transfect mMSCs. Cells were plated at 50% confluence on fibronectin coated circular cover glass attached to punched petri-plates. 2.5 µg of siRNA was diluted in 150 µl of OptiMEM (solution A) and 12.5 µl of Dharma FECT (ThermoScientific) was diluted in 150 µl of OptiMEM (solution B). Solutions were incubated for 5 minutes at room temperature. Solution B was gently mixed into solution A (hereafter, referred to as transfection mixture) and incubated at room temperature for 30 minutes. Cells were washed with OptiMEM (Invitrogen) after removing the growth media. Transfection mixture was added drop wise to the cell plate and shaken gently. An additional 1.6 ml of OptiMEM medium is added and the cells were incubated at 37°C. Transfection medium was replaced with complete growth medium ( -MEM; Invitrogen) after 5 hours. Transfected cells were imaged for nuclear and cell projected areas after 48 hrs of transfection.
8
Lipofectamine Transfection of ASOs
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Cultured cells were seeded out one day prior to transfection to allow the cells to adhere. After 24 h the cells were washed once in Opti-MEM (Gibco) and transfected using a mixture of Lipofectamine 2000 (5 µg/mL (Sk-Hep-1), 10 µg/mL (Hep3B), 10 µg/mL (HEK293) 10 µg/mL (THLE-3) ThermoFisher), Opti-MEM and the ASOs at a final concentration of 1, 5 or 25 nM. Four hours after transfection, the mixture was carefully removed from the cells and fresh medium was added to each well. HepG2 cells were reverse-transfected. A mixture of Lipofectamine 2000 (2.5 µg/mL, ThermoFisher), Opti-MEM combined with the ASOs at a final concentration of 1, 5 or 25 nM were incubated at RT, and the HepG2 cells were added after 15 min of incubation. Knockdown was assessed 48 h after transfection. The cells were harvested, total RNA isolated and RNA expression measured using RT-qPCR as described.
In the experiments in which the effect of PURPL knockdown was assesses in combination with Doxorubicin or Nutlin treatment, cells were transfected as described above with ASOs at a final concentration of 25 nM for 24 h before DMSO (0.5%), Doxorubicin (Sigma; 5 µM) or Nutlin (Sigma; 5 µM) was added to the appropriate wells. Both Doxorubin and Nutlin were reconstituted in DMSO (Sigma).
In the experiments in which the effect of PURPL knockdown was assesses in combination with Doxorubicin or Nutlin treatment, cells were transfected as described above with ASOs at a final concentration of 25 nM for 24 h before DMSO (0.5%), Doxorubicin (Sigma; 5 µM) or Nutlin (Sigma; 5 µM) was added to the appropriate wells. Both Doxorubin and Nutlin were reconstituted in DMSO (Sigma).
9
Transient Transfection Protocols for Cell Lines
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For transient transfection of HeLa-M, COS-7, HEK293T, A549, and HaCaTs, plasmid DNA was complexed with FuGENE 6 (Promega, E269A) at a 1:3 ratio in 100μL Opti-MEM (Gibco, 31985–070). After a 10 min incubation, the transfection solution was added to cells, which were imaged 18–36h later. siRNA transfection was performed using 40 nM siRNA complexed with Lipofectamine RNAiMAX in Opti-MEM (ThermoFisher). Cells were imaged ~48h post-transfection. For transfection of iPSCs, 2μg plasmid DNA was incubated with 10 μL Lipofectamine Stem (Thermo Fisher) and 200μL OptiMEM for 10m and added dropwise to the dish. Cells were imaged 24h after transfection. Neurons were transfected at two days in vitro (DIV2) with 1μg total DNA (0.5 μg Mito-dsRed2, 0.5 μg Lifeact-EGFP) and 1ul Lipofectamine 2000 (Invitrogen) for 30 min and imaged 16h later.
10
Transient Transfection Protocols for Cell Lines
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