PNT2 cells were transfected with the four HOXB13 expression vectors (Wt and mutated) and with the control vector (Entry) using TurboFectin 8.0™ transfection reagent (Origene). Briefly, one day before transfection, PNT2 cells were plated in 60mm dishes at a density of 1.5×105 in complete growth medium, in order to reach a confluence of approximately 60% at the day of transfection. For each transformation, 250μL of Opti-MEM medium (GIBCO®) were mixed with TurboFectin 8.0™ and incubated at room temperature for five minutes. The vector DNA was added and incubated at room temperature for 30 minutes. In order to achieve the best transfection efficiency, three different ratios of TurboFectin:DNA were used for each vector: 3:1 (7.5μL of TurboFectin for 2.5μg of DNA), 3:2 (7.5μL of TurboFectin for 5μg of DNA) and 6:1 (15μL of TurboFectin for 2.5μg of DNA). The TurboFectin:DNA containing mixtures were then added dropwise to the cells and incubated for 48 hours at regular growth conditions. At this point, cells were exposed to selective pressure by adding regular growth medium supplemented with 450µg/µL of G418 (GIBCO®). Four to five weeks later stable cell populations were obtained.
Turbofectin 8.0 transfection reagent
Manufactured by OriGene
Sourced in United States
TurboFectin 8.0 is a transfection reagent designed for the efficient delivery of nucleic acids into a variety of mammalian cell lines. It is a cationic lipid-based formulation that forms complexes with DNA, RNA, or other molecules to facilitate their uptake by cells.
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Lab products found in correlation
Mmp 3 sirna, by Thermo Fisher Scientific (2 mentions)
Lenti x concentrator, by Takara Bio (2 mentions)
One shot stbl3 chemically competent cells, by Thermo Fisher Scientific (2 mentions)
Lenti concentrator, by OriGene (2 mentions)
Lenti x gostix plus, by Takara Bio (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Zymopure plasmid maxiprep kit, by Zymo Research (2 mentions)
Opti mem medium, by Thermo Fisher Scientific (1 mentions)
Sitran 1.0 transfection reagent, by OriGene (1 mentions)
Two well chamber slides, by BD (1 mentions)
Alexa 555 conjugated anti mouse secondary antibody, by Cell Signaling Technology (1 mentions)
Dm4000b, by Leica (1 mentions)
Secrete pair dual luminescence assay kit, by GeneCopoeia (1 mentions)
Opti dmem, by Thermo Fisher Scientific (1 mentions)
Pvector control vector, by OriGene (1 mentions)
4d nucleofector, by Lonza (1 mentions)
Opti mem 1, by Thermo Fisher Scientific (1 mentions)
Puromycin, by Merck Group (1 mentions)
Pgfp 5 rs, by OriGene (1 mentions)
15 protocols using turbofectin 8.0 transfection reagent
1
Stable Transfection of HOXB13 in PNT2 Cells
2
Caveolin-1 Modulation in Alveolar Epithelial Cells
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Primary cultured AEC-II were seeded onto Transwell Clear inserts and cultured to 60–70 % confluence before transfection. For Cav-1-siRNA transfection, cells were transfected with 10 nm of a rat Cav-1-specific siRNA (5’-GGCAUAGCACAAGUAAUAGUCUGTA-3’; Cat.# SR500200; OriGene Technologies, Rockville, MD, USA) or 10 nm of a non-silencing scrambled control siRNA (Cat.#SR30004;OriGene Technologies) using a siTran 1.0 transfection reagent (OriGene Technologies), according to the manufacturer’s protocol. For Cav-1-cDNA transfection, cells were exposed to hyperoxia (85 % O2 and 5 % CO2) and transfected with Cav-1 cDNA plasmid (1 μg/mL; Cat.#RR200414; OriGene Technologies) or with an empty vector plasmid (1 μg/mL; Cat.#PS100001; OriGene Technologies) as control using Turbofectin 8.0 transfection reagent (OriGene Technologies), following the manufacturer’s instructions.
Cav-1 mRNA and protein expression levels were monitored using reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively, to determine the best concentration of Cav-1-siRNA or Cav-1 cDNA plasmid for transfection and the best post-transfection time for the lowest and highest Cav-1 expression. Cells transfected with siRNA or cDNA plasmid were harvested at 48 and 72 h after transfection, respectively, before further experiments.
Cav-1 mRNA and protein expression levels were monitored using reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively, to determine the best concentration of Cav-1-siRNA or Cav-1 cDNA plasmid for transfection and the best post-transfection time for the lowest and highest Cav-1 expression. Cells transfected with siRNA or cDNA plasmid were harvested at 48 and 72 h after transfection, respectively, before further experiments.
3
Stable Knockdown of PGC1α in Pancreatic Cancer Cells
4
Confirming H7HA Gene Expression by Indirect Immunofluorescence
5
Transfection and Nano-CuO Exposure Protocol
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Transfection was performed as described in our previous study with modifications [13 (link)]. Briefly, BEAS-2B cells were seeded in the inserts and incubated for 12 h, then U937* cells were seeded at a ratio of 1:1 or 9:1 (BEAS-2B:U937*) onto the BEAS-2B cell layer in antibiotic-free medium supplemented with 10% FBS and allowed to attach. Cells were then transfected with a mixture of 6 µL of TurboFectin 8.0 Transfection Reagent (Origene, Rockville, MD, USA) and 30 nM of MMP-3 siRNA (Ambion, Carlsbad, CA, USA) in a total volume of 1 mL antibiotic-free and FBS-free medium for 6 h. Afterward, 1 mL medium containing 2 times FBS and antibiotics was added, and the cells were incubated for another 12 h. Silencer™ Select Negative Control No. 2 siRNA (Ambion, Carlsbad, CA, USA) was used as a negative control. After exposure to Nano-CuO for 12 h, the conditioned media were collected and the roles of MMP-3 in the cleavage of OPN and activation of fibroblasts were determined.
6
miR-375 Regulation of CCND2 3'UTR
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A reporter construct containing a binding site at CCND2 3′UTR for miR-375 (GeneCopoeia, Rockville, MD, USA) was introduced into PC-3 cells using Turbofectin 8.0 transfection reagent (Origene, Rockville, MD, USA). A vector without CCND2 3′UTR (GeneCopoeia) was used as experiment control. Vectors were co-transfected along with pre-miR-375 as described. Luciferase activity was assessed with the Secrete-Pair™ Dual Luminescence Assay Kit (GeneCopoeia, Rockville, MD, USA) according to the manufacturer’s instructions. Experiments were performed in triplicates at 48 and 72 h following co-transfection. At 72 h, miR-375 levels were measured by RT-qPCR to confirm its forced or silenced expression.
7
Nano-CuO Induced EMT and MMP-3 Silencing
8
Generation of Dnaic2 Knockdown Mice
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Dnaic2 KD mice were generated as described by Zhang et al.22 Briefly, we first isolated and then cultured female germline stem cells (FGSCs) from ovaries of CD‐1 mice in the short‐term. FGSCs were then cultured for 3‐5 days and were transfected with the pRS‐U6‐shDnaic2‐SV40‐Puro vector using TurboFectin 8.0 transfection reagent according to the manufacturer's instructions (OriGene). After 48 h, the FGSCs were transplanted into ovaries of recipients sterilised by chemotherapy. The recipient mice were mated to wild‐type mice. The genotypes were then identified by PCR and Southern blotting. PCR was performed using 1 μg genomic DNA from mouse tails with primer set (sense: 5′‐TCGACCCTGTGGAATGTGT‐3′; anti‐sense: 5′‐GGGCTTGTACTCGGTCAT‐3′) specific for SV40 early promoter. Southern blotting was used to confirm the results of Dnaic2 KD mice, and was performed according to the method mentioned above.22 Finally, western blotting was carried out using rabbit anti‐DNAI2 or anti‐β‐tubulin (see below).
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Dnaic2 KD mice were generated as described by Zhang et al.
9
CRISPR-Mediated Vaccinia Virus Engineering
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A total of 2 × 106 CV-1 cells were seeded in a 6-well plate a day before transfection. Cells at 60–70% confluency were transfected with 1 µg each of plasmid encoding Cas9HFc and gRNA using 6 µL of TurboFectin 8.0 transfection reagent (Origene Technologies, Rockville, MD, USA) in 250 µL of opti-DMEM (Thermo Fisher Scientific, Waltham, MA, USA). At 24 h post-transfection, cells were infected with CAL1 VACV at an MOI of 0.02 in high glucose DMEM supplemented with 2% FBS. Two hours after viral infection, the cells were washed with PBS. 1.5 mL DMEM was added to the wells and the plate was incubated at 37 °C with CO2 for 30 min before being transfected with 2 µg of the donor plasmid generated above. The cells were further incubated at 37 °C with 5% CO2 and in a humidified atmosphere for 24 h. The mixture of infected/transfected cells was harvested and stored at −80 °C until the virus was screened and purified.
10
Silencing PRDX3 in Breast Cancer Cells
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MDA-MB-231 cells were transfected with shRNA targeting PRDX3 (shPRDX3) or scrambled shRNA cassette (sh-NT) using Turbofectin 8.0 transfection reagent (Origene Technologies, Inc., Rockville, MD, USA), according to the manufacturer’s instruction. Cells were then selected by puromycin dihydrochloride. siRNA transient transfection was conducted in BT-549 cells using a targeting pool of 4 siRNAs from Dharmacon (Chicago, IL, USA) as described previously [21 (link)].
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