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. 2023 Nov 17;4(4):102709. doi: 10.1016/j.xpro.2023.102709

Figure 1.

Figure 1

Multicolor antigen density quantification panel using pre-calibrated beads alongside monoclonal antibodies of known fluorophore-to-protein ratios

(A) Entire 9-color antibody panel containing six antibodies specific for neuroblastoma cell surface markers used for antigen density quantitation and three additional colors for gating purposes.

(B) Fluorescent calibration beads contain several populations with known fluorophore molecules/bead and allow conversion of MFI into antigen density for antibody-fluorophore conjugates with known F:P ratio used at saturating concentrations.

(C) Plate set up for titration of antibodies used in the panel to assess saturating concentrations using the negative control cell line CHO-K1 and two neuroblastoma specimens (the cell line SMS-SAN and the patient-derived neuroblastoma cells ST16-4045). Serial dilutions range from no antibody to 0.1, 1, 5, 10, 20 and 30 μg/mL in a total of 100 μL FACS buffer containing fixable viability dye. Example plate setup for three antibodies (GPC2-PE, GD2-BV510, ALK-APC) is shown. A second plate with the same setup was run for B7H3-PE-Cy7, L1CAM-BV421, NCAM-BV605).