Human truestain fcx

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.

1 × 10⁵ tumor cells per well were cultured in 96-well plates (flat bottom plate for adherent cells, round bottom plate for suspension cells) at 37 °C 5% CO₂. 7C6 (a human IgG1 mAb kindly provided by Dr Kai W. Wucherpfennig) or human IgG1 isotype control (Biolegend, Koblenz, Germany) antibody were added at 10 µg/mL. After 24 h of culture, MICA and MICB on cell surface were detected following staining with APC conjugated anti-MICA/B antibody or IgG2a isotype control. For detaching adherent cells without disturbing the integrity of surface molecule, Accutase (Biolegend, Koblenz, Germany) was used. Prior to the staining process, Fc receptors were blocked with Human TrueStain FcX™ (Biolegend, Koblenz, Germany) at a final dilution of 1:100. Hoechst 33258 (Cayman Chemical, Hamburg, Germany) was added before flow cytometry analysis for viable cells gating. Samples were acquired using FACS Canto II (BD).