PE/Cyanine7 anti-mouse CD252 (OX40L) Antibody

Protocol tips

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single-cell suspensions from fresh mouse CLNs or cultured BM-macrophages were filtered by a 70-μm cell strainer, centrifuged, and resuspended. Cells were incubated with a stain kit (LIVE/DEAD Fixable Yellow Dead Cell Stain Kit, L34959, Thermo Fisher Scientific, Waltham, MA)

Upstream tips
single-cell suspensions from fresh mouse CLNs or cultured BM-macrophages were filtered by a 70-μm cell strainer, centrifuged, and resuspended. Cells were incubated with a stain kit (LIVE/DEAD Fixable Yellow Dead Cell Stain Kit, L34959, Thermo Fisher Scientific, Waltham, MA)

Publication protocol

"Flow cytometry was performed as previous reported methods.38–40 In brief, single-cell suspensions from fresh mouse CLNs or cultured BM-macrophages were filtered by a 70-μm cell strainer, centrifuged, and resuspended. Cells were incubated with a stain kit (LIVE/DEAD Fixable Yellow Dead Cell Stain Kit, L34959, Thermo Fisher Scientific, Waltham, MA), followed by staining with anti-CD11b-APC (clone M1/70; eBioscience, San Diego, CA), anti-F4/80-APC/Cy7 (clone BM8; Biolegend), anti-TSLPR-PE (FAB5461P; R&D Systems, Minneapolis, MN), and anti-OX40L-PE/Cy7 (clone RM134L; Biolegend).

The gating strategy used in this study was as follows: dead cells were excluded by gating live dye negative cells, subsequently gated on the basis of forward scatter height versus forward scatter area (singlets 1), then gated on side scatter height versus side scatter area (singlets 2). Cells were then gated on F4/80+CD11b+ cells, and frequencies of F4/80+TSLPR+ and F4/80+ OX40L+ were recorded. Negative controls consisted of FMO cells. A BD Canto II Benchtop cytometer (Becton Dickinson, San Jose, CA) was used for flow cytometry, and data were analyzed with BD Diva software v6.7 (BD Biosciences, San Jose, CA) and FlowJo software v10. (Tree Star Inc., Ashland, OR)."

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