Cells were first stained with fixable viability dye (ThermoFisher Scientific, Paisley, UK), followed by staining with specific antibodies. Extracellular staining was performed in phosphate-buffered saline plus 0.1% bovine serum albumin and 0.05% sodium azide; intracellular staining was performed using fix/perm solution and permeabilization buffer (eBioscience, Hatfield, UK) as per the manufacturer’s protocol. Two percent mouse serum was added to cells to block nonspecific staining before addition of antibodies. The following antibodies were used in this study: anti-CD1c (clone L161); anti-CD3 (clone OKT3 and clone UCHT1); anti-CD4 (clone RPA-T4); anti-CD11c (clone 3.9); anti-CD14 (clone M5E2); anti-CD15 (clone W6D3); anti-CD16 (clone 3G8); anti-CD19 (clone HIB19); anti-CD20 (clone 2H7); anti-CD25 (clone BC96); anti-CD45 (clone Hl30); anti-CD45RA (clone HI100); anti-CD56 (clone MEM-188); anti-CD86 (clone IT2.2); anti-FOXP3 (clone 259D); anti-HLA-DR (clone L243); anti-SIRPα (clone SE5A5) (all from Biolegend, London, UK); anti-CD141 (clone 1A4, BD and clone M80, R&D Systems, Minneapolis, MN, USA); and anti-CD103 (clone B-Ly7, eBioscience). Production of the anti-integrin β8 (clone ADWA16) is described below.
Cells were analyzed using an LSR Fortessa or LSRII (BD, Oxford, UK), and data were analyzed using Flowjo software (Flowjo, OR, Ashland).
Cells were analyzed using an LSR Fortessa or LSRII (BD, Oxford, UK), and data were analyzed using Flowjo software (Flowjo, OR, Ashland).