Immulon 2hb flat bottom plates

For Vero E6 binding studies, cells were grown to confluence in 48 well plates. Media was replaced with DMEM supplemented with 2% FBS and the indicated compounds. Cells were incubated at 10°C (preventing internalization and entry) until equilibrated and SARS-CoV-2 was added at MOI 5. Plates were returned to 10°C for 1 hour. Media was then removed and cells were washed three times with cold DPBS (GIBCO, Cat# 14190144), removing any unbound virus. Then 0.05% Trypsin-EDTA (GIBCO, Cat# 25300054) was added to control wells for 5 minutes at 37°C, and washed. After the final wash, all media was removed and replaced with TRIzol (Invitrogen, Cat# NCC1701D). RNA was isolated and analyzed as described. Binding studies in HEK 293T cells were performed using rVSV-SARS-CoV-2 Spike virions (rVSV/Spike) and binding was performed at room temperature in IMMULON 2HB flat bottom plates (Thermo Scientific, Waltham, MA).

Immulon 2 HB flat-bottom plates (96-well; Thermo Fisher Scientific) were coated with 200 μL of 1 × 10⁶ TCID₅₀/mL of ZIKV in Opti-MEM I medium (Gibco). For ZIKV protein binding assay, plates were incubated with recombinant mLILRB4 or hLILRB4 proteins, followed by primary antibodies against target proteins overnight at 4°C. For antibody titer assay, plates were incubated with serum samples overnight. The signals were read with VICTOR X Light Luminescence Plate Reader (PerkinElmer) at 450 nm.

rMMRN1 binding to immobilized human FG (±pre‐treatment with 0.2 U/mL thrombin to induce fibrin formation), FN, or triple‐helical collagen peptides was evaluated based on methods described,⁶ using Immulon 2 HB flat‐bottom plates (Thermo Fisher Scientific) coated (overnight, 4°C) with 1 µg/well protein or peptide.