For fluorescence staining, PBMC were fixed in ice‐cold Cytofix/Cytoperm solution (BD Biosciences), washed in cold PBS/0.5% bovine serum albumin (Aurion, Wageningen, NL) containing 0.5% Saponin (Sigma) and labelled with Hoechst 33342 (1/10000; Thermo Fisher Scientific), Annexin V FITC, PE‐conjugated mouse anti‐CD3 mAb (IgG1; BD Biosciences), unconjugated mouse anti‐CD33 mAb (IgG1; BD Biosciences) as well as unconjugated rabbit anti‐PU.1 (IgG; Santa Cruz Biotechnology). Binding of unlabeled antibodies was detected using a biotinylated goat anti‐mouse IgG1 mAb (1/250; Dianova) in combination with PE‐TexasRed‐conjugated streptavidin (1/100;Thermo Fisher Scientific) and a Cy5‐conjugated donkey anti‐rabbit IgG (1/250; Dianova) as secondary antibodies respectively. PBMO were identified as Annexin− CD33+ CD3− cells.
Image files were automatically acquired in flow with an ImageStream imaging cytometer (Amnis, Seattle, WA). Single color controls were used to calculate the spectral crosstalk matrix. Compensated image files were analyzed with IDEAS 3.0 (Amnis). The expression of PU.1 or CD33, respectively, was calculated by the addition of the intensity values of all pixels in the respective image.
Image files were automatically acquired in flow with an ImageStream imaging cytometer (Amnis, Seattle, WA). Single color controls were used to calculate the spectral crosstalk matrix. Compensated image files were analyzed with IDEAS 3.0 (Amnis). The expression of PU.1 or CD33, respectively, was calculated by the addition of the intensity values of all pixels in the respective image.