Cflow plus program

Single and double immunofluorescence staining were used to determine the subpopulations of lymphocytes, and their expression of the NKG2D receptor, MICA/B, and the DNAM‐1 receptor. mAbs for the following markers were used: CD19, CD4, CD8, CD56, CD16, CD161, NKG2D, MICA, MICB, DNAM‐1, NKG2D/CD56, NKG2D/CD8, DNAM‐1/CD56, DNAM‐1/CD8. In brief, three hundred thousand cells/well, suspended in PBS containing .2 % BSA and .02% NaN3, were incubated with appropriate concentrations of primary mAbs/Abs for 30 min with gentle shaking. After wash, the cells were incubated with FITC‐ and/or PE‐conjugated antibodies for 30 min in darkness. Isotype‐matched primary IgG antibodies (DAKO) were used in negative controls. CD45/CD14 staining was used for lymphocyte gating. Ten thousand events per marker were collected by Accuri 6C Flow Cytometer (BD Biosciences) and analyzed with CFlow Plus program (BD Biosciences).

Receptor downregulation of NKG2D expression with exosomes was done as described.¹³ In brief, PBMCs were incubated in 5 % CO₂, at 37°C for 24 h in absence/presence of equal concentration (40 μg/ml) of native OC‐exosomes isolated from HGSC patient serum, or after treatment with specific mAbs against NKG2D ligands or CD63. After incubation, the cells were stained for NKG2D receptor expression; the events were collected by Accuri 6C Flow Cytometer (BD Biosciences) and analyzed in CFlow Plus program (BD Biosciences) with a gate on CD56⁺/CD3⁻ NK‐cells.