Flow Cytometry Cell Surface Binding

Cells were detached from tissue culture flasks using CellStripper dissociation reagent (Corning), quenched with PBS with calcium and magnesium (PBS^+/+) supplemented to contain 2% FBS, and centrifuged at 1,500 × g at 4°C for 5 min. Cells (5 × 10⁵ cells per sample) were stained with human anti-HS (1:750; Amsbio 370255-S), human anti-CS (1:750; Sigma C8035), human anti-Mxra8 (1 μg/ml; MBL International W040-3), or mouse anti-Mxra8 (1 μg/ml; 4E7.D10 [31 (link)]) antibodies at 4°C for 1 h. Cells were incubated with Alexa Fluor 647 antibody (1:1,000; Thermo Fischer Scientific) at 4°C for 1 h. Samples were washed twice with VDB between incubations. Samples were fixed with 1% PFA at 4°C for 5 min and analyzed by flow cytometry (LSRII flow cytometer; BD Biosciences). Binding events were gated using secondary-antibody-only control samples as the no-binding controls, and median fluorescent intensity (MFI) was determined using FlowJo V10 software.