Fitc conjugated anti np antibody

Cells were washed with 0.1% BSA in PBS, followed by specific surface staining with 1:50 APC-conjugated anti-CD14 (immunotools, Friesoythe, Germany) and 1:20 PE-conjugated anti-CD20 (BD biosciences, CA, USA) at 4°C for 30 min. Cells were then washed and fixed with 4% paraformaldehyde at 4°C for 30 min. Subsequently, cells were stained for surface α2,3 SA with 1:100 biotinylated Maackia Amurensis Lectin I (MAL I) (Vector Laboratories, CA, USA) at 4°C for 30 min. After washing, cells were incubated with 1:200 Per-CP Streptavidin (BD Biosciences, CA, USA). For intracellular staining of viral NP, cells were permeabilized in Cytofix/Cytoperm reagent (BD Biosciences, CA, USA) at 4°C for 10 min, then washed with Perm/wash (BD Biosciences, CA, USA), incubated with 1:500 FITC-conjugated anti-NP antibody (Millipore, USA) at 4°C for 30 min, washed and suspended in 2% paraformaldehyde. To detect α2,3 SA expression for single stain, cells were stained with 1:100 FITC-conjugated MAL I (Vector Laboratories, CA, USA). Stained cells were detected using a CytoFLEX flow cytometry (Beckman Coulter, USA), and the data were analyzed using Kaluza Analysis Software—Version 2.0. (Beckman Coulter, USA). A total of 10,000 cells were acquired for each flow cytometry analysis.

Cells were washed and fixed with 4% paraformaldehyde at 4 °C for 30 min. Subsequently, cells were permeabilized in Cytofix/Cytoperm reagent (BD Biosciences, San Jose, CA, USA) at 4 °C for 10 min, then washed with perm/wash (BD Biosciences, San Jose, CA, USA). Cells were incubated with 1:500 FITC-conjugated anti-NP antibody (Millipore, Burlington, MA, USA) at 4 °C for 30 min, washed and suspended in 2% paraformaldehyde. Stained cells were detected using CytoFLEX flow cytometry (Beckman Coulter, Brea, CA, USA), and the data were analyzed using Kaluza Analysis software version 2.0. (Beckman Coulter, Brea, CA, USA).