GLuc mRNA was labeled with Cy5 using a Label IT Cy5 Labeling Kit (Mirus Bio, Madison, WI, USA). Huh-7 cells were seeded in a 6-well plate (100,000 cells/well) and incubated for 24 h. To each well, Cy5-labeled mRNA (Cy5-mRNA)-loaded PICs were added (250 ng of mRNA/well). After 24 h incubation, the transfected cells were washed twice with cold PBS and collected after trypsinization. The cells were centrifuged and resuspended in PBS. The Cy5 intensity of the cells treated with Cy5-mRNA was measured in a BD LSR II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA), equipped with a 633 nm laser and 660/20 nm filter.
Label it cy5 labeling kit
Manufactured by Mirus Bio
Sourced in United States
The Label IT Cy5 Labeling Kit is a product designed for labeling nucleic acids, such as DNA and RNA, with the Cy5 fluorescent dye. The kit provides the necessary reagents and protocols to enable efficient and reliable labeling of target molecules. It is intended for use in various applications that require fluorescent detection or analysis of nucleic acids.
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4 protocols using label it cy5 labeling kit
1
Cy5-labeled mRNA Transfection in Huh-7 Cells
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GLuc mRNA was labeled with Cy5 using a Label IT Cy5 Labeling Kit (Mirus Bio, Madison, WI, USA). Huh-7 cells were seeded in a 6-well plate (100,000 cells/well) and incubated for 24 h. To each well, Cy5-labeled mRNA (Cy5-mRNA)-loaded PICs were added (250 ng of mRNA/well). After 24 h incubation, the transfected cells were washed twice with cold PBS and collected after trypsinization. The cells were centrifuged and resuspended in PBS. The Cy5 intensity of the cells treated with Cy5-mRNA was measured in a BD LSR II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA), equipped with a 633 nm laser and 660/20 nm filter.
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GLuc mRNA was labeled with Cy5 using a Label IT Cy5 Labeling Kit (Mirus Bio, Madison, WI, USA). Huh-7 cells were seeded in a 6-well plate (100,000 cells/well) and incubated for 24 h. To each well, Cy5-labeled mRNA (Cy5-mRNA)-loaded PICs were added (250 ng of mRNA/well). After 24 h incubation, the transfected cells were washed twice with cold PBS and collected after trypsinization. The cells were centrifuged and resuspended in PBS. The Cy5 intensity of the cells treated with Cy5-mRNA was measured in a BD LSR II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA), equipped with a 633 nm laser and 660/20 nm filter.
2
Fluorescent Labeling of eIF4E and mRNA
3
Cy5-labeled DNA Uptake in APP/PS1-N2a Cells
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Following the manufacturer’s instructions, pDNA was labeled using a Label IT Cy5 labeling kit (Mirus, Madison, WI, USA). In brief, APP/PS1-N2a cells (5 × 104/well) were seeded into a 35 mm confocal culture dish. After 24 h, the CA-PEI CDs (5 μL, 5 mg/mL) were incubated with Cy5-labeled DNA (0.1 mg DNA/well, the optimal sample mass ratio) in an incubator at 37 °C for 30 min. The resulting complex was added to the cell culture medium and incubated for an additional 24 h at 37 °C. Subsequently, the cells were washed with 1 mL of PBS, and fluorescence images were captured using a laser scanning confocal microscope (FV3000, Olympus, Tokyo, Japan).
4
Labeling and Binding of Poly(I:C) RNA
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Synthetic double stranded RNA Poly (I∶C) (Sigma) was labeled with Label IT Cy5 labeling kit (Mirus). Briefly, 5 µg of RNA was incubated at a 1∶1 (v∶w) ratio of Label IT Cy5 reagent to nucleic acid. 40 ng of labeled Poly(I∶C) was incubated with 20 ng GFP-PA2G4 fusion protein in binding buffer (50 mM Tris pH 7.4, 0.5 mM EDTA, and 150 mM NaCl) at room temperature for 45 min. GFP-PA2G4 was immunoprecipitated with anti-GFP magnetic beads as previously described [20] (link). An aliquot of gel loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol, 50% glycerol) was added to the reaction mixture and resolved on 10% non-denaturing polyacrylamide gels in 1× TBE. Gels were scanned on a Typhoon FLA 9500 Imager (GE Healthcare) using 633/670 nm for Cy5 filter and 489/508 nm for GFP filter.
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