Fluorescent labelling of EV RNA was performed by incubating EVs (1 μg in 160 μl) with 1.6 μl of 1 μM SYTO RNASelect reagent (Thermo Scientific, USA) according to manufacturer’s instructions. Unincorporated dye was removed using MW3000 exosome purification spin columns (Thermo Scientific, USA). Suspension of labelled EVs (100 ng) was added to culture media of A549 cells growing on cover slips in wells of 24-well plate and incubated for 16h. Culture media was then removed, cells were washed twice with cold PBS and fixed for 15 min with cold 4% PFA in PBS followed by two washes with cold PBS. Cover slips were mounted on glass sides and nuclei were counter-stained with DAPI using ProLong Diamond Antifade Mountant (Thermo Scientific, USA). Fluorescent signal in the cells was visualized on ZEISS LSM 510 META Confocal Microscope.
Syto rnaselect reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
The SYTO RNASelect reagent is a fluorescent nucleic acid stain used for the detection and quantification of RNA in cells and tissues. It is designed to selectively bind to RNA, providing a reliable method for visualizing and analyzing RNA content in various biological samples.
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Lab products found in correlation
Lsm 510 meta confocal microscope, by Zeiss (1 mentions)
Prolong diamond antifade mountant, by Thermo Fisher Scientific (1 mentions)
Click it rna alexa fluor 594 imaging kit, by Thermo Fisher Scientific (1 mentions)
1 step human coupled ivt kit, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated anti rabbit secondary antibody 7074s, by Cell Signaling Technology (1 mentions)
1 6 hexanediol, by Tokyo Chemical Industry (1 mentions)
2 protocols using syto rnaselect reagent
1
Fluorescent Labeling of EV RNA Uptake
2
RNA Labeling and Imaging Workflow
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1-Step Human Coupled IVT kit, poly-rA RNA, Click-iT RNA Alexa Fluor 594 imaging kit, and SYTO-RNASelect reagent were purchased from Thermo Fisher Scientific. Recombinant human IPO7 (Ag27257), recombinant human GIT1 (Ag10658), rabbit polyclonal anti-NAP1L4 antibody (16018-1-AP), rabbit polyclonal anti-PAF1 antibody (15441-1-AP), rabbit polyclonal anti-IPO7 antibody (28289-1-AP), rabbit polyclonal anti-RPL5 antibody (15430-1-AP), and rabbit polyclonal anti-EIF3A antibody (11423-1-AP) were purchased from Proteintech. Horseradish peroxidase–conjugated anti-rabbit secondary antibody (#7074S) was purchased from Cell Signaling. Chambered coverglasses (uncoated or hydrophilic polymer MAS-coated) were obtained from Matsunami Glass. 1,6-Hexanediol was purchased from Tokyo Chemical Industry.
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