Cd14 pe clone mφp9

Blood samples were processed within 10 hours after collection, stained with monoclonal antibodies for 15 min at room temperature in the dark and further diluted in central inlet buffer. The following directly conjugated monoclonal antibodies were used in this study: CD3-APC (clone HIT3a), CD14-PE (clone MφP9), CD19 FITC (clone HIB19), CD45-PerCP (clone 2D1), CD61-PE (clone VI-PL2), CD66b FITC (clone G10F5) and CD235a APC (clone GA-R2), as well as matched isotype controls (all from BD Bioscience, San Jose, CA, USA). Stained samples were analyzed before and after separation on a FACSCanto II flow cytometer (BD Bioscience) and acquired data was further analyzed using the FlowJo software (Tree Star Inc., Ashland, OR, USA).

To investigate the cell types of HBoV1 persistence, three populations were isolated sequentially from HBoV1 DNA-positive tonsils (Prep 3) using CD antibody-coupled magnetic beads (Dynabeads; Invitrogen), according to the manufacturer’s instructions: T cells with anti-CD3 beads, B cells with anti-CD19 beads for the CD3-depleted cells, and monocytes with anti-CD14 beads for the CD3-CD19-depleted cells. To test the purity of the three isolates, cells from the beads were released with DETACHaBEADS (Invitrogen), and labeled with anti-human CD19-FITC (CD19 labeled with fluorescein isothiocyanate) (clone SJ25-C1; Invitrogen), CD3-PE/Cy5.5 (CD3 labeled with phycoerythrin or Cy5.5) (clone UCHT1; Abcam), or CD14-PE (clone MφP9; BD Biosciences) and analyzed on a BD Accuri C6 flow cytometer as described previously (26 (link)). Assessment of the cell fraction proportions is described in Text S1.