Human PRRX1b (NM_022716.4) was cloned into a retroviral pZOME vector carrying the puromycin resistance gene. 5 µg of constructs were transfected with plasmids carrying vsvg and gagpol into 293et cells using Transfectin transfection reagent (Bio-Rad). 48 h after transfection, the virus particles were harvest by filtering the medium through 0.45 µm syringe filter and were stored at -80ºC. The day before infection, mouse 3T3-L1 cells were plated into 6-well plates. The next day the medium was removed and virus-containing medium was added in the presence of polybrene (4 µg/ml). Two days after infection the cells successfully transduced with virus were selected with puromycin (2 µg/ml). A comparable control cell line expressing green fluorescence protein (GFP) was also generated.
Transfectin transfection reagent
Manufactured by Bio-Rad
Transfectin is a lipid-based transfection reagent developed by Bio-Rad for the delivery of nucleic acids, such as plasmid DNA, siRNA, or mRNA, into eukaryotic cells. It facilitates the uptake of these molecules by the cells, enabling their expression or silencing.
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Lab products found in correlation
3 protocols using transfectin transfection reagent
1
Overexpression of PRRX1b in 3T3-L1 Cells
2
Measuring Intracellular cAMP Dynamics
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Cells were transiently transfected with the Epac – based sensor for cAMP (Ponsioen et al., 2004 (link)), using Transfectin transfection reagent (Biorad). After transfection, neurons were maintained in culture for an additional 10 - 12 h before FRET imaging experiments to allow the genetically encoded sensor to be expressed. Imaging experiments were performed on an inverted microscope Olympus IX 70 with PlanApo 60X NA 1.4 oil-immersion objective. Excitation at 430 nm was performed with a Polychrome IV monochromator (Till Photonics GmbH, Germany) equipped with a 150 W xenon lamp. Images were captured every 3 s with a 16-bit sCMOS pco.edge camera (pco imaging), and the emission wavelengths were separated with a dual - emission beam splitter (Multispec Microimager; Optical Insights) with a 505 nm dichroic filter and 480 ± 15 and 545 ± 20 nm emission filters for CFP and YFP, respectively. All filters and dichroics were from Chroma Technology. The system is controlled by a custom-made software. Exposure time was set to 200-300 ms. Data were processed offline with ImageJ software (National Institutes of Health).
FRET changes were measured as changes in the background - subtracted 480/545 nm fluorescence emission intensities on excitation at 430 nm and expressed as R/R0, where R is the ratio at time t and R0 is the ratio at time = 0 s.
FRET changes were measured as changes in the background - subtracted 480/545 nm fluorescence emission intensities on excitation at 430 nm and expressed as R/R0, where R is the ratio at time t and R0 is the ratio at time = 0 s.
3
Modulation of Secretase Activities
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HEK293T cells and SH-SY5Y cells were grown in an incubator at 37 °C and 5% CO2. The HEK293T growth medium was DMEM (PAA) supplemented with 10% FCS (PAA), 2 mM glutamine and 1 mM sodium pyruvate. SH-SY5Y cells were grown in DMEM/Ham’s F12 (Biochrom) supplemented with 10% FCS (PAA), 2 mM glutamine, 1 mM sodium pyruvate and 1× non-essential amino acids (PAA). Cells were transfected with the indicated plasmids using polyethylenimine or Transfectin transfection reagent (BioRad). The cell culture medium was exchanged 18 hours after transfection. The newly added medium contained the indicated inhibitors and was conditioned for 6 hours. Activity of following secretases was inhibited by specific inhibitors from Calbiochem: α-secretase (GM6001; 50 µM), β-secretase (β-secretase inhibitor IV; 20 µM) and γ-secretase (L-685,458; DAPT; γ-secretase inhibitor I and III; each at 10 µM).
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