Lsr fortessa 4 laser flow cytometer

After stimulation, human neutrophils were washed and stained with fluorochrome-conjugated mouse anti-human Abs (BD Bioscience) targeting surface antigens including CD11b -Alexa Fluor 700, CD62L - BV510, CD66b - PerCP-Cy5.5, CD49d - BB515, CD16 - V450, and CD15 -RPE as well as the intracellular activation marker p38 MAPK - pT180/pY182 - RPE. Live dead cells were also measured using APC -LIVE/DEAD™ staining (ThermoFisher Scientific). The median fluorescence intensity for each marker was determined using the LSR Fortessa 4-laser flow cytometer (BD Biosciences).

LLC MK2 cells were inoculated with hMPV A2-GFP (CAN97-83) or hMPV B2 (TN/83–1211) in the presence of 1 µg/mL TPCK-trypsin. Seventy-two hours after inoculation, the cells were detached, washed, and blocked in 1% BSA. Cells were stained with 1 µg/mL sdHMPV16 or Ctrl sdAb. Afterward, the cells were washed and treated with the fixation/permeabilization solution kit according to the manufacturer’s protocol (catalog no. 555028; BD). After fixation and permeabilization, cells were stained with monoclonal mouse anti-human metapneumovirus antibody (1/500, catalog no. MAB8510; Merck/Millipore). For sdAb conditions, cells were also stained with a monoclonal rabbit anti-histidine antibody (1/1,000; catalog no. PA1-983B; ThermoScientific). The binding of primary antibodies was revealed with goat anti-mouse IgG coupled to Alexa Fluor 647 (1/600; Invitrogen), donkey anti-rabbit IgG coupled to Alexa Fluor 488 (1/600; Invitrogen), and goat anti-human IgG coupled to Alexa Fluor 594 (1/600; Invitrogen), followed by analysis on an LSR Fortessa 4 laser flow cytometer (BD). Contour plots were created with the FlowJo software.