Quantibrite pe beads

For FACS analysis adherent cells at about 70% confluence were detached using 0.1 mM EDTA in PBS (Sigma Aldrich, Darmstads, Germany), centrifuged and re-suspended in PBS containing 0.2% BSA. Cell aliquots (2.5 × 10⁵ cells) were treated with primary monoclonal antibody PE conjugate, (Millipore, Burlington, MA, USA) or isotype control (Santa Cruz Biotechnology, Dallas, TX, USA), at the same concentration (14 µg/mL), whole a reaction volume of 50 µL for 30 min at 4 °C. After washing, the cells were analyzed by using a flow cytometer equipped with a 488 nm argon laser (FACScan, Becton Dickinson, Franklin Lakes, NJ, USA). A total of 20,000 events per sample were collected. Integrin quantification with Quantibrite PE beads (BectonDickinson Biosciences) was evaluated as previously described [46 (link)]. Values of fluorescence intensity were obtained from the histogram statistic of CellQuest software.

Variance in flow cytometric measurements was determined from a 5 × 3 × 3 experiment, testing the effect of the donor (biological variability), instrument (technical variability), and day (environmental variability) on measured fluorescence intensities. PB was collected from 5 healthy donors on 3 non-consecutive days. The samples were stained with the 4C MRD panel and analyzed on three different instruments. The antibody binding capacity of the PE-tagged targets CD56, CD22, CD38, and CD79b was determined from the consecutive measurements of three of the five donors using Quantibrite PE beads (BD BioSciences, Heidelberg, Germany) according to the manufacturer’s instructions. The MFIs for different populations were used to evaluate the effect of the selected parameter on overall variability using the eta squared value from ANOVA. F-statistics were determined to test models using only the donor as a factor against models using all factors.

Expression of human leucocyte antigen-antigen D-related (HLA-DR) on CD14⁺ monocytes was measured by flow cytometry according to the manufacturer’s recommendations (Quantibrite HLA-DR/Monocyte antibody cocktail, BD Bioscience, Heidelberg, Germany). Briefly, 50 μL of EDTA-anticoagulated whole blood was incubated with 20 μl of the antibody cocktail and incubated for 30 min. Afterwards, erythrocyte lysis was performed by adding 450 μl FACS Lysing solution (BD Bioscience, Heidelberg, Germany) and further incubation for 15 minutes. Measurement was immediately done on a FACSCalibur flow cytometer (BD Bioscience). In addition, a 4-point calibration curve (Quantibrite PE Beads, BD Bioscience) was also measured daily to enable the transformation of the measured sample values to molecules of HLA-DR.

To quantify the expression of HLA-DR on monocyte surface, flow cytometry (FACSVerse^™ flow cytometer, BD Bioscience, Heidelberg, Germany) was conducted after incubating 50 μl ETDA-anti-coagulated whole blood with 20 μl anti-HLA-DR antibody (Quantibrite anti-HLA-DR/Monocyte antibody, BD Bioscience, Heidelberg, Germany) and after lysis of erythrocytes (FACS Lysing solution BD Bioscience, Heidelberg, Germany) as previously described and according to manufacturer`s instructions [18 (link)]. HLA-DR surface expression was measured as median fluorescence intensity after gating out monocytes based on CD14 expression (S10 Fig).
For quantification of HLA-DR molecules on cell surface (`counts per monocyte´), determined sample values were converted using daily measured 4-point calibration curves (Quantibrite PE Beads, BD Bioscience, Heidelberg, Germany).

Tumor line CD33 surface expression was determined by flow cytometry. The anti-CD33 antibody clones AC104.3E3 (Miltenyi Biotec), WM53 and HIM3-4 (both from BioLegend) were used. Isotype-controls were used for negative gating. Estimation of CD33 site density on cell lines of interest was performed using QuantiBRITE PE beads (BD Biosciences, San Jose, CA) using the antibodies bound per cell (ABC) method as per manufacturer's protocol. Beads conjugated with PE molecules at four different intensities were used to generate a standard curve, and tumor cells stained with anti CD33 antibodies conjugated to PE beads were acquired under identical settings. The intensity of PE staining for each cell line was then used to extrapolate the number of PE molecules per tumor cell.