Colonic LP samples (2 million cells/staining) were stained at 4°C for 20 min with the following antibodies: Ly6C-FITC (AL-21, BD Pharmingen), F4/80-PE (BM8, BioLegend), Siglec F-PE-CF594 (E50-2440, BD Bioscience), Ly6G PE-Cy7 (1A8, BioLegend), CD11b-APC (M1/70, eBioscience), CD11c-AF700 or -APC-R700 (N418, BioLegend), MHCII-BV421 (M5/114.15.2, BioLegend), and CD45-BV510 (30-F11, BioLegend) at 4°C for 20 min. Dead cells were identified by staining with eFluor 780 Fixable Viability dye (eBioscience) and gatings were performed as described previously (22 (link)).
For analyses of integrin receptors on human monocytes, mouse Hoxb8 monocytes and mouse BMDMs, antibodies to integrins β1 (mouse 9EG7 (23 (link))/human P5D5) (24 (link)), α6 (GoH3, BD Pharmingen), α5 (mouse 5H10-27, BD Pharmingen/human P1D6) (24 (link)), α4 (PS/2, Abcam), α3 (mouse polyclonal, R&D/human P1B5) (24 (link)), β3 (mouse 2C9.G2, Biolegend/human B3A, Chemicon), and β2 (mouse C71/16, BD Pharmingen/human TS1/18, Thermo Scientific) were employed. Cells were analyzed with a Gallios (Beckman Coulter) or Celesta (BD) flow cytometer and FlowJo software.
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