A total of 105 cells were incubated with a flow cytometry media (PBS w/o Ca2+ or Mg2+ with 0.01% sodium azide and 1% fetal bovine serum) enriched with human TrueStainFcX™ (BioLegend, San Diego, CA, USA) for 15 min at 4°C in the dark. Cells were then incubated with the antibodies for 30 min at 4°C in the dark, with additional mixing at 15 min. Cells were washed twice in FACS media and re-suspended in 100 µl of 1% FlowFix (Polysciences, Warrington, PA, USA). The following antibodies were employed: CD1a (HI149; BioLegend, San Diego, CA, USA), CD14 (Tuk4; Invitrogen, Grand Island, NY, USA), CD83 (HB15e; BD, San Jose, CA, USA), CD115 (AFS98; BioLegend, San Diego, CA, USA), CD124 (G077F6; BioLegend, San Diego, CA, USA), and CD126 (UV4; BioLegend, San Diego, CA, USA).
The frequency of naturally occurring DC was analyzed with a BDCA enumeration kit (Myltenyi Biotec, Germany) (15 (link), 28 (link)). Enumeration protocol takes into account leukocytes count in the blood.
Cells were analyzed with an LSR™ (BD, San Jose, CA, USA) or a FACSCalibur™ (BD, San Jose, CA, USA). Non-specific antibodies were used as a negative control. At least 10 × 104 were collected. Data were expressed as a frequency of cells while mean fluorescent intensity (MFI) was a measure of receptor density. MFI is presented as relative to non-specific binding after exposing cells to non-specific antibodies.
The frequency of naturally occurring DC was analyzed with a BDCA enumeration kit (Myltenyi Biotec, Germany) (15 (link), 28 (link)). Enumeration protocol takes into account leukocytes count in the blood.
Cells were analyzed with an LSR™ (BD, San Jose, CA, USA) or a FACSCalibur™ (BD, San Jose, CA, USA). Non-specific antibodies were used as a negative control. At least 10 × 104 were collected. Data were expressed as a frequency of cells while mean fluorescent intensity (MFI) was a measure of receptor density. MFI is presented as relative to non-specific binding after exposing cells to non-specific antibodies.