HuH7-H1 cells were maintained in DMEM-F12 (Gibco) media supplemented with 5% FBS (Gibco), 1x Penicillin-Streptomycin (Sigma), 600 μg/ml of G418 (Clontech) at 37 °C and 5% CO2. Protein transfections were performed with 50 ng of capsid protein with the Xfect™ Protein Transfection kit (Clontech) according to the manufacturer’s protocol. After transfection, cells were incubated for 4 h at 37 °C, washed with PBS, fixed with 4% paraformaldehyde, and permeabilized with 0.2% Triton X-100 before labeling with a 1:500 dilution of the Mab3120 monoclonal anti-Cp antibodies (Institute of Immunology Co., Ltd.), and then probed with a 1:500 dilution of AlexaFluor594-conjugated goat anti-mouse secondary antibody (Thermo Fisher Scientific). Cells were visualized with a Leica TCS SP8 confocal microscope equipped with Leica HyD Detector, a 63X (f1.4 numerical aperture) oil immersion objective, and appropriate lasers for exciting DAPI, BoDIPY, and AlexaFluor594. A detailed description of transfection and sample preparation is available in Supporting information .
Xfect protein transfection kit
Manufactured by Takara Bio
Sourced in Japan
The Xfect™ Protein Transfection kit is a laboratory tool designed for efficient delivery of proteins into mammalian cells. It provides a simplified protocol for transfection of active proteins, enzymes, or antibodies into a variety of cell types.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 594 conjugated goat anti mouse secondary antibody, by Thermo Fisher Scientific (1 mentions)
Hyd detector, by Leica camera (1 mentions)
Tcs sp8 confocal microscope, by Leica camera (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
μ dishes, by Ibidi (1 mentions)
4 protocols using xfect protein transfection kit
1
Protein Transfection and Immunofluorescence Imaging
2
Protein Transfection of SCs
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GFP antibody or CPEB1 antibody was transfected into SCs using the X-fect Protein Transfection Kit (Clontech) according to the manufacturer’s instructions.
3
Xfect Protein Transfection for Peptide Delivery
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The Xfect Protein Transfection kit (Takara Bio Inc., Shiga, Japan) was used to introduce the peptides into cells according to the manufacturer’s instructions. Briefly, cells were grown to 60–70 % confluence in coverslip-like-bottomed 35-mm diameter culture dishes (μ-Dishes; ibidi, Verona, WI, USA) and incubated at 4 °C for 1 h in serum-free medium supplemented with 0.25 × Xfect protein transfection reagent containing 0.4 μg (for RLE-6TN cells) or 0.2 μg (for the other cells) of either the C-ICD or C-ICDmut peptide labeled with FITC or unlabeled, as indicated. The cells were washed with serum-free medium and incubated in growth medium. The same treatment was applied 24 h later. The cells were subjected to Western blot analyses after an additional 24 h, and mitochondrial labeling or terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays were conducted.
4
RIG-I Activation Assay in 293T Cells
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293 T cells were transfected with reporter genes (0.5 µg of p-55C1BLuc and 25 ng of p-RL-TK)1 (link),11 (link). After 16 h, naïve or dissociated RIG-I, isolated from MDA5 KO 293 T cells, was transfected into cells using the Xfect protein transfection kit (Takara) for 4 h. Cells were further cultivated for 4 h in fresh medium. Luciferase activities were determined as previously described9 (link).
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