A volume of 300 µL of cells were seeded onto round glass coverslips (12 mm diameter) in 24-well plates at a density of 105 cells per well and incubated overnight at 37°C and 5% CO2. A suspension of fluorescein isothiocyanate (FITC)-loaded NPs in SF and SR phenol red-free DMEM achieving a final NPs’ concentration of 250 µg/mL was added after aspiration of the old medium and washing the cells with phosphate-buffered saline (PBS) to remove any debris. After the predefined incubation times (3 and 24 h), the medium was aspirated, cells were washed twice with cold PBS, and the cell membrane was stained with wheat germ agglutinin-AlexaFluor 594 (WGA-AF594) (Invitrogen, Germany) according to the manufacturer’s instructions. Cells were then washed with cold PBS twice again, fixed with 4% paraformaldehyde (PFA) (Elektron Technology, UK) and the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI blue) (Sigma-Aldrich, Germany). The coverslips were then lifted, dried and mounted on glass slides using Mowiol mounting medium (Sigma-Aldrich, Germany) for the preservation of the fluorescence intensity and imaged using a Zeiss Axio star Plus fluorescence microscope (Carl Zeiss, Sweden).38 (link)
Wga af594
Manufactured by Thermo Fisher Scientific
Sourced in Germany
The WGA-AF594 is a fluorescently labeled wheat germ agglutinin (WGA) conjugate. WGA is a lectin that binds to N-acetylglucosamine and sialic acid residues, which are commonly found on the extracellular surface of cells. The WGA-AF594 conjugate can be used to label and visualize these cell surface features.
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2 protocols using wga af594
1
Nanoparticle Internalization in Cells
2
Multicolor Fluorescence Imaging of Cells
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Images were recorded using a Leica TCS SP8 confocal microscope. GFP imaging was done with an excitation at 488 nm and detection of the emitted light between 500–540 nm. FM4-64 (Molecular Probes, Karlsruhe, Germany) staining was carried out for 5 min after addition of 1 μl of a 2 mM stock solution dissolved in dimethylsulphoxide per 1 ml of culture and imaging was done using an excitation wavelength of 561 nm and detection between 580–660 nm. For wheat germ agglutinin-Alexa Fluor 594 (WGA-AF594, Invitrogen) 2 μl of a 1 mg ml−1 stock solution (dissolved in PBS) was used in a volume of 0.5 ml (excitation at 561 nm detection at 610–650 nm).
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