Him3 4

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.

Cells (5 × 10⁴ cells) were cultured onto poly-l-lysin-coated round coverslips for 24 h. Coverslips were then fixed with 4% PFA for 10 min at RT. Cell spots were then blocked with 50 μL of PBS (incl. 2% FBS and 2 mM EDTA) for 10 min before adding anti-human CD33-FITC (1/200, eBioscience, HIM3-4) and anti-human TLR2-PE (1/50, BioLegend, clone QA16A01) antibodies for 2 h at RT. Cell spots were then washed in PBS and analyzed on a Zeiss LSM 700 confocal microcope.

Cells (5 × 10⁵ cells) were washed and re-suspended in 100 μL of PBS (incl. 2% FBS and 2 mM EDTA) containing 20 μL of FcR Blocking Reagent (Miltenyi) for 10 min at 4 °C. Cells were then stained with anti-human CD33-FITC (1/200, eBioscience, HIM3-4) and anti-human TLR2-PE (1/50, BioLegend, clone QA16A01) antibodies for 20 min at 4 °C. Cells were then washed twice and acquired on a FACSCalibur (BD) and analyzed using FlowJo software (Tree Star, Inc.).