DC-SIGN-expressing HEK 293 (DC-HEK) cells, as reported by Lang et al. (31 (link)) were grown in DMEM-F12 (Life Technologies, UK) containing 10% v/v FCS and blasticidin (5 µg/mL) (Gibco). The cells were grown on 13 mm glass cover slips till a monolayer of cells was formed and then incubated with 15 µg/mL of recombinant ghA, ghB, and ghC (MBP as a negative control) separately in serum free medium and left to incubate for 30 min in 37°C. Cells were washed with PBS and fixed using 4% v/v paraformaldehyde for 10 min, rinsed again with PBS three times, and then blocked with 5% FCS for 30 min. The slides were incubated for 30 min with mouse anti-MBP antibody to detect MBP fusion proteins and rabbit anti-DC-SIGN antibody to reveal expression of DC-SIGN in DC-HEK cells. After three washes for 30 min each and incubation with secondary antibodies: Alexa Fluor 568 conjugated goat anti-mouse antibody (Thermo Fisher) and Alexa Fluor 488 conjugated goat anti-rabbit antibody (Abcam) for 30 min, the slides were then washed in PBS, mounted, and observed under Leica DM4000 Fluorescent microscope using Leica Application Suite.
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