Sulfo nhs lc lc biotin

Recombinant SUDV GP was biotinylated and coupled to 1 µm FITC⁺ Neutravidin beads (Life Technologies). Briefly, SUDV GP was biotinylated using Sulfo-NHS-LC-LC biotin (ThermoFisher Scientific), and excess biotin was removed using a Zeba desalting column (ThermoFisher Scientific). Biotinylated SUDV GP was incubated with Neutravidin-coated fluorescent beads (ThermoFisher Scientific) at 4 °C overnight (1 µg biotinylated protein per 1 µl of beads). Beads were washed twice with 0.1% BSA in PBS and resuspended in 100 µl of 0.1% BSA in PBS per 1 µl of coupled beads. Sera from vaccinated guinea pigs were diluted 1:100 in cell culture medium and incubated with GP-coated beads for 2 h at 37 °C. Freshly isolated neutrophils from donor blood were added at a concentration of 5.0 ×10⁴ cells per well and incubated for 1 h at 37 °C. Cells were stained at 1:100 with CD66b (Pacific Blue, Clone G10F5; Biolegend cat # 305111), fixed with 4% paraformaldehyde, and analyzed by flow cytometry on a Sartorius iQue flow cytometer, and a minimum of 30,000 events were recorded and analyzed. Neutrophils were defined as SSC-A^high CD66b⁺. The phagocytic score was determined using the following formula: (percentage of FITC⁺ cells) x (median fluorescent intensity (MFI) of the FITC⁺ cells)/10,000.

Petri dishes were coated with poly-l-lysine before seeding and transfection. Two days after transfection, the cells were washed with PBS-CM (100 ml 10x PBS, 1 mM MgCl₂, 0.5 mM CaCl₂, pH 8.0 adjusted with NaOH, Milli-Q until total volume of 1000 ml) followed by adding 0.5 mg/ml Sulfo-NHS-LC-LC-biotin (Thermo fisher scientific, Rockford, USA). After 30 min incubation, cells were washed with 0.1% (w/v) PBS-CM BSA and PBS. Cells were then lysed with lysis buffer, and protein concentration was measured with a BCA test. Input samples were taken to be able to check transfection efficiency by performing Western blot. Then, lysates with the same amount of proteins (as calculated with the BCA test) were added with the neutravidin agarose beads (Thermo fisher scientific, Rockford, USA) and incubated overnight rotated at 4 °C. After incubation, protein lysates were washed with lysis buffer and Laemmli and DTT buffer were added and incubated at 37 °C for 30 min before loading the protein samples on SDS-PAGE.