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The following day, the cells were transfected using Lipofectamine 2000 with the AAV vector along with AAV-DJ and AAV-pHelper packaging vectors (Cell Biolabs) at a ratio of 1:1:1. The next day, fresh DMEM media with 10% FBS was added; 48 h later, the cells and media were collected and subjected to four freeze-thaw cycles by alternating between ...

Lipofectamine had the highest efficiencies. Metafectene Pro had less toxicity than Lipofectamine. Increasing the amount of Metafectene Pro (1:4) increases toxicity but not the transfection efficiency significantly. Increasing the post-transfection period increases toxicity: at 48 h the toxicity of Metafectene Pro and Lipofectamine is high.
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e angiogenesis and proliferation. However, the underlying mechanisms of miR-145 in tumor angiogenesis of UM are still not well illustrated. Thus, we aimed to explore the potential target genes or pathways regulated by miR-145 in UM and the effect of miR-145 on invasion and angiogenesis. Methods Totally, 24 choroid samples were collected in our study, including 12 UM samples and 12 normal uveal ...
The present invention relates to a method for transducing a target cell, the method comprising the step of contacting a target cell with a retroviral vector and a poloxamer having a molecular weight of 12,8 kDa to about 15 kDa.
Sep 19, 2018 · The serotonin-1A (5-HT1A) receptor is a key regulator of serotonergic activity and is implicated in mood and emotion. However, its post-transcriptional regulation has never been studied in humans. In the present study, we show that the “intronless” human 5-HT1A gene ( HTR1A ) is alternatively spliced in its 3′-UTR, yielding two novel splice variants. These variants lack a ∼1.6 kb ...
Lipofectamine had the highest efficiencies. Metafectene Pro had less toxicity than Lipofectamine. Increasing the amount of Metafectene Pro (1:4) increases toxicity but not the transfection efficiency significantly. Increasing the post-transfection period increases toxicity: at 48 h the toxicity of Metafectene Pro and Lipofectamine is high.
Aug 05, 2016 · The S6 Kinase (S6K) proteins are some of the main downstream effectors of the mammalian Target Of Rapamycin (mTOR) and act as key regulators of protein synthesis and cell growth. S6K is overexpressed in a variety of human tumors and is correlated to poor prognosis in prostate cancer. Due to the current urgency to identify factors involved in prostate cancer progression, we aimed to reveal the ...
We studied the function of osteoblast cadherins in vivo by transgenic expression of a truncated N-cadherin with dominant-negative action, driven by an osteoblast-specific promoter (OG2-NcadΔC). During the first 3 months of life, bone mineral density was reduced, whereas percent body fat was increased in transgenic animals compared with wild-type littermates, with associated decreased bone ...
Jul 29, 2020 · Lipofectamine 2000 Transfection Reagent (Invitrogen, 11,668,019) was used to transfect the PANC-1, ASPC-1 and SW1990 cell lines with the miR-619-5p mimics, inhibitors and siRNAs, and the expression vector of PVT1, Pygo2 or ATG14 was transfected using Lipofectamine 3000 (Invitrogen, L3000015) according to the manufacturer’s protocols.
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  • HEK293 vs HEK293T - which is better for transfection (reply: 2) Stable miRNA transfection - (reply: 7) ... Cell death with DMSO treatment, during transfection using polybrene - (reply: 2) ... Lipofectamine transfection under the microscope - ...
  • Make sure to use the polybrene-containing media to make the cell solution in this step. To seed the cells: Prepare a batch of cells as follows: Dilute 350,000 cells into a total volume of 7 mL of DMEM complete + 10 µg/mL polybrene. Mix well by pipetting or inverting the tube. Aliquot 1 mL of cell suspension (i.e., 50,000 cells) into each well ...
  • 2. Mix 8 µl Polybrene (1 mg/ml aliquot) with 957 µl culture. 3. Add 35 µl of lentiviral particles and mix the solution by flicking the tube. The final concentration of Polybrene is 8 µg/ml. 4. Remove culture medium from the cells, and add the Lentivirus/Polybrene mixture. 5. Optionally, perform a spinoculation step. Centrifuge for 90 min at ...
  • Nov 06, 2019 · To transfect siRNAs, 50 pmol of pre-annealed siRNA duplex in OptiMem transfection medium were mixed with Lipofectamine RNAiMAX reagent (Invitrogen) according to manufacturer’s protocol and then added to 293T cells seeded in a 12-well plate in 1 mL of growth medium for 6 h. All siRNAs used in this work are listed in Additional file 1: Table S2.
  • Lipofectamine® 3000 reagent is designed to efficiently transfect difficult-to-transfect cells, yielding superior transfection performance across the broadest array of cell types. Successfully transfected cell types include: Fibroblast (3T3, COS-7) Myoblast (C2C12, L6 CRL-1458)

Jun 17, 2020 · Lentiviral vectors expressing control shRNA (non-target) and G9A shRNA constructs (Dharmacon, USA) were transfected into 293T using Lipofectamine 3000 according to the manufacturer’s instructions. After 48 h of transfection, the supernatants containing lentivirus were harvested and used, to infect target cells with 6 μg/ml polybrene (Sigma ...

Mar 01, 2003 · The plasmids were transiently transfected into Phoenix-E cells using LipofectAmine Plus (Life Technologies Inc., Rockville, Maryland, USA). Infection of BMMs. We isolated BMMs from bone marrow of 4- to 8-week-old mice, cultured them overnight in α-MEM containing 10% heat-inactivated FBS, and subjected them to Ficoll-Hypaque gradient ... Aug 25, 2020 · Pancreatic cancer is one of the most lethal human cancers. N6-methyladenosine (m6A), a common eukaryotic mRNA modification, plays critical roles in both physiological and pathological processes. However, its role in pancreatic cancer remains elusive. LC/MS was used to profile m6A levels in pancreatic cancer and normal tissues. Bioinformatics analysis, real-time PCR, immunohistochemistry, and ...
Make sure to use the polybrene-containing media to make the cell solution in this step. To seed the cells: Prepare a batch of cells as follows: Dilute 350,000 cells into a total volume of 7 mL of DMEM complete + 10 µg/mL polybrene. Mix well by pipetting or inverting the tube. Aliquot 1 mL of cell suspension (i.e., 50,000 cells) into each well ...

Jul 17, 2020 · Cell transfection was conducted using Lipofectamine 3000 Transfection Reagent (Invitrogen) according to the manufacturer’s instructions. In brief, HEK293 cells were seeded to be 70–90% confluent at transfection. For 12-well plate/10 cm dish transfection, 1–1.5 μg/16 μg plasmids were used.

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Oct 18, 2013 · Polybrene is widely used in viral gene transduction. Polybrene has no cytotoxic activity at low concentrations, but negatively affects the proliferation of corneal keratinocytes at concentrations greater than 10 μg/ml . It also negatively impacts the proliferation of human mesenchymal stem cells, even if used at 1 μg/ml .