Expression and binding assay of recombinant Siglecs

 Seed 293T cells to a T75 flask at ~ 50 % confluence a day before the transfection. The flask should be near-confluent the next day.

 Filter-sterilize the plasmid (30 μg) solution using Spin-X (12,000 g, 5 min). Mix 3 mL Opti-MEM and 75 μL LipofectAMINE 2000 in a 15 mL tube and leave for 5 min at room temperature. Add the filter-sterilized plasmid into the tube and mix well. Leave for 20 min at room temperature, and add evenly to the semi-confluent flask of 293T cells. Culture overnight in CO₂ incubator.

 Remove medium, gently wash once with 5 mL Opti-MEM or PBS, and add 15 mL of Opti-MEM + 2% low IgG-FCS. Culture for 3 days in CO₂ incubator.

 Transfer culture sup from the flask (by decantation) to 50 mL tube. Add 15 mL fresh Opti-MEM + 2% low IgG-FCS to the flask and put back into CO₂ incubator, and culture for 3 days. Meanwhile, centrifuge the collected culture sup (at 1,000–1,500 g, 5 min) to remove debris. Store at 4˚C.

 Collect culture sup from the flask into 50 mL tube. Centrifuge the collected culture sup (1,000–1,500 g, 5 min) to remove debris. Culture sup harvested on day 3 and day 6 may be combined.

 Add 1/50 vol. of 1 M Tris-HCl buffer to the clarified culture sup. Add 0.1 mL (as packed gel) protein A-Sepharose, prepared as instructed by the manufacturer. Incubate overnight at 4˚C, with gentle agitation.

 Prepare disposable column assembly. Pour the culture sup + protein A-Sepharose into the column. Wait until all culture sup pass through the column.

 Add 2 mL Tris-buffered saline to the column and drain (= "wash the column"). Wash the column once again with 2 mL Tris-buffered saline.

 Wash the column with 1 mL of 20 mM HEPES-NaOH buffer. Cap the column tip and add the same buffer (final volume should be ~ 500 μL). Add 10 mU of sialidase and incubate at 37˚C for 1 h. Remove the bottom cap and wash the column 5 times with 1 mL Tris-buffered saline, to remove sialidase.

 Wash the column twice with 1 mL of 0.1 M citrate-NaOH buffer, pH 5.8.

 Elute Siglec-Fc protein from the column twice with 1 mL 0.1 M glycine-HCl buffer, pH 3.0, and collect the eluate. Immediately add 0.2 mL of 1 M Tris-HCl buffer to neutralize. The protein A-Sepharose column can be regenerated by washing with 1 mL of 6 M guanidine HCl, followed by wash with Tris-buffered saline.

 Transfer the neutralized eluate to ultrafiltraion device and concentrate. Discard flow-through. Replace to a desired buffer by repeated concentration and buffer addition, if necessary. Recover the Siglec-Fc fusion protein and analyze quantity (by protein assay) and quality (by SDS-PAGE). Store the protein at –20˚C.

 Prepare 5 μg/mL solution of protein A in immobilization buffer, and add 100 μL/well to the wells of 96-well plate. Each Siglec – probe combination should be assayed in triplicate wells. Incubate overnight at 4˚C.

 Remove protein A solution. Pat the plate lightly against paper towel to remove excess solution lingering to the wells. Add 150 μL ELISA buffer to each well, then revert to discard the solution, and pat lightly against paper towel (= "wash the plate"). Wash the plate once again.

 Add 150 μL ELISA buffer to each well and incubate at room temperature for 1 h. Meanwhile, prepare 1–5 μg/mL solution of Siglec-Fc or human IgG in ELISA buffer.

 Remove the ELISA buffer and lightly pat the plate against paper towel. Add 100 μL Siglec-Fc (or human IgG) solution in each well. Incubate at room temperature for 2 h.

 Remove Siglec-Fc solution by aspiration, and wash the plate 3 times with 150 μL/well ELISA buffer. Add 100 μL/well of 1–10 μg/mL glycan-PAA-Bio probes, diluted in ELISA buffer. Incubate at room temperature for 2 h.

 Remove probe solution by aspiration, and wash the plate 3 times with 150 μL/well ELISA buffer. Dilute streptavidin-alkaline phosphatase at 1:1,000 in ELISA buffer. Add 100 μL/well. Incubate at room temperature for 1 h.

 Remove streptavidin-alkaline phosphatase solution, and wash the plate 3 times with 150 μL/well ELISA buffer.

 Add 100 μL/well alkaline phosphatase substrate and incubate at room temperature for 5 min to overnight. Measure absorbance at 405 nm using a plate reader. Check the plate at 5 min, 15 min, 30 min, 1 h, and 2 h after addition of the substrate.

 Harvest mammalian cells (e.g., BEAS-2B) to be stained. Trypsinization may be better avoided. Approximately 0.1–1 × 10⁶ cells/sample should be used. Wash cells with PBS.

 Suspend cells in sialidase buffer (at 1–5 × 10⁶/mL), aliquote to 2 tubes and add sialidase to one of the tubes (at 50 mU/mL). Incubate at 37˚C for 30 min. Centrifuge to remove supernatant, and wash with PBS for 3 to 5 times to remove sialidase.

 Suspend cells in staining buffer (at 1–5 × 10⁶/mL) and aliquote at 0.1 mL/tube. Add 1 μg Siglec-Fc and 0.5 μg biotinylated anti-FLAG antibody per tube. Mix and incubate on ice for 1 h.

 Add 0.9 mL of staining buffer, centrifuge and remove supernatant.

 Suspend cells in 0.1 mL staining buffer containing 1 μL of streptavidin-conjugated R-phycoerythrin (= 1:100 dilution). Mix and incubate on ice for 30 min.

 Add 0.9 mL of staining buffer, centrifuge and remove supernatant.

 Suspend in 0.5 mL of staining buffer, filter through Cell-Strainer or nylon mesh if necessary (e.g., if the cells are clumped), and analyze with a flow cytometer.