Quickcal program

Receptor density studies were performed by flow cytometry on a MACSQuant VYB (Miltenyl Biotec) essentially as described20 (link). Briefly, anti-EGFR GA201 and anti-HER2 trastuzumab IgGs were first labeled with Alexa Fluor 647 labeling kit (Invitrogen) according to the supplier’s recommendations. Antibody concentration and fluorochrome to protein (F:P) ratio were determined by a ND-1000 spectrophotometer (NanoDrop). Parental NCI-H358 and NCI-H358.HER2.ko cells at ~4 × 10⁶ cells/mL were washed with ice-cold FACS Buffer (PBS pH 7.2, 2% FBS, 2 mM EDTA and 0.1% sodium azide) followed by incubation with the conjugated antibodies at saturating concentrations (≥ 20 μg/mL) for 30 min at 4 °C. After washing with FACS buffer, cells were fixed in ice-cold 1.8% paraformaldehyde (PFA) and detection of bound antibodies was performed on MACSQuant VYB using MACSQuantify™ software. Results were analyzed using the FlowJo analysis software (Tree Star). For quantitation of EGFR and HER2 receptor density on cells, Quantum Alexa Fluor 647 MESF (Molecules of Equivalent Soluble Fluorochrome) beads (Bangs Laboratories) were analyzed on the flow cytometer using similar settings to establish a standard curve. Using the QuickCal program (Bangs Laboratories) the calculated MESF was then divided by the antibody F:P ratio to give a corrected Antibody Binding Capacity (ABC).

Receptor density studies were performed by flow cytometry on a MACSQuant VYB (Miltenyl Biotec) essentially as described [27 (link)]. Briefly, anti-CD4 (ibalizumab), anti-EGFR (GA201) and anti-HER2 (B1D2) IgGs were first labeled with Alexa Fluor 647 labeling kit (Invitrogen) according to the manufacturer’s instructions. Antibody concentration and fluorochrome to protein (F:P) ratio were calculated by a ND-1000 spectrophotomer (NanoDrop). Cells at ~ 4 × 10⁶ cells/mL were first washed with ice-cold FACS Buffer (PBS pH 7.2, 2% FBS, 2 mM EDTA and 0.1% sodium azide) followed by incubation with saturating concentration (≥ 20 μg/mL) of conjugated antibodies for 30 min at 4°C. After washing with FACS buffer, cells were fixed in ice-cold 1.8% paraformaldehyde (PFA) and detection of bound antibodies was determined on MACSQuant VYB using MACSQuantify™ software. Data were analyzed with the FlowJo analysis software. For quantitation of CD4, EGFR and HER2 density on cells, Quantum Alexa Fluor 647 MESF (Molecules of Equivalent Soluble Fluorochrome) beads (Bangs Laboratories) were processed on the flow cytometer using similar settings. QuickCal program (Bangs Laboratories) was used to establish a standard curve. The calculated MESF was then divided by the antibody F:P ratio to give a corrected Antibody Binding Capacity (ABC).