Chemotx system plate

Recombinant human CXCL12β (3 nM), alone or in combination with purified recombinant proteins were placed in the lower chamber of a 96-well ChemoTx System plate (Neuro Probe # 101–5) in RPMI 1640 1% FBS. As internal controls within each assay, medium or recombinant protein alone were used. MonoMac-1 cells (1.25 × 10⁵) were placed on the upper compartment and separated from the lower chamber by a 5 μm pore size filter. The cells were incubated at 37° C for 3 h in a humidified incubator with 5% CO₂. The migrated cells in the lower chamber were stained with 5 μl of CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega # G3580) for 2.5 h at 37° C with 5% CO₂, measuring absorbance at 490 nm using a Synergy H1 plate reader (Bio-Tek). The following recombinant proteins were used: SARS-CoV-2 N-His (Sino Biological # 40588-V07E), HCoV-OC43 N-His (Sino Biological # 40643-V07E), GFP-His (Thermo Fisher # A42613), NL63 N-His (Sino Biological # 40641-V07E), 229E N-His (Sino Biological # 40640-V07E), and HKU1 N-His (Sino Biological # 40642-V07E).

Recombinant human CXCL12β (3 nM), alone or in combination with purified recombinant proteins, was placed in the lower chamber of a 96-well ChemoTx System plate (Neuro Probe, nos. 101-3 and 101-5) in RPMI 1640 containing 1% FBS. As internal controls within each assay, medium or recombinant protein alone was used. PBMCs, MonoMac-1, and MOLT-4 cells (1.25 × 10⁵) were placed on the upper compartment and separated from the lower chamber by a 3- or 5-μm pore size filter. The cells were incubated at 37°C for 3 hours in a humidified incubator with 5% CO₂. The migrated cells in the lower chamber were stained with 5 μl of CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, no. G3580) for 2 hours at 37°C with 5% CO₂, measuring absorbance at 490 nm using a Synergy H1 plate reader (Bio-Tek).
The following recombinant proteins were used: SARS-CoV-2 S1-His (Sino Biological, no. 40591-V08B1), SARS-CoV-2 N-His (Sino Biological, no. 40588-V08B), SARS-CoV-2 N-His (Acro Biosystems, no. NUN-C5227), SARS-CoV-2 N-His (Ray Biotech, no. 230-30164), SARS-CoV-1 N-His (Acro Biosystems, no. NUN-S5229), and MERS-CoV N-His (Acro Biosystems, no. NUN-M52H5). SARS-CoV-2 N-His from Sino Biological (no. 40588-V08B) was used in all assays unless indicated.