Anti cd14 percp cy5.5 m5e2

To define the tropism of each poxvirus for mononuclear cell subsets, PBMCs were seeded at 4 × 10⁵ cells/well in Roswell Park Memorial Institute (RPMI)‐1640 medium (Sigma) supplemented with 10% heat‐inactivated FCS, 2 mM L‐glutamine and 40 µg mL⁻¹ gentamycin in round‐bottom 96‐well plates and infected with recombinant PCPV‐GFP, MVA‐GFP or VACV‐GFP at a MOI of 1. The next day, cells were washed with PBS, incubated with FcR Blocking Reagent (Miltenyi Biotec) diluted 1/10 and subjected to surface staining with either anti‐lineage (CD3/19/20/56)‐APC (UCHT1, HIB19, 2H7, 5.1H11; BioLegend, San Diego, CA, USA), anti‐CD14‐PerCP‐Cy5.5 (M5E2; BD Pharmingen, San Diego, CA, USA) and anti‐CD16‐BV605 (3G8; BioLegend) or anti‐CD3‐PE‐Vio770 (REA613; Miltenyi Biotec), anti‐CD4‐APC (A161A1; BioLegend), anti‐CD56‐BV421 (5.1H11; BioLegend) and anti‐CD19‐AF700 (HIB19; BioLegend) antibodies in combination with LIVE/DEAD^TM near‐IR fluorescent reactive dye (Molecular Probes, Eugene, OR, USA). Samples were run on a MACSQuant 16 flow cytometer (Miltenyi Biotec) and analysed using the KALUZA 1.3 software (Beckman Coulter, Life Sciences, Villepinte, France). The percentage of infected cells (GFP⁺) within live monocytes (Lin^–CD14⁺CD16^‐/bright), and CD4⁺ T (CD3⁺CD56⁻CD4⁺), CD8⁺ T (CD3⁺CD56⁻CD4⁻), NK (CD3⁻CD56⁺) and B (CD3⁻CD56⁻CD19⁺) singlets was determined.