Flow Cytometry for Anti-DCC [IPI-mDCC.114]

EC₅₀: A fourteen point titration of antibody concentrations, ranging from 660 nM (0.1 mg/mL) to 4.42 pM with a 1:2.5 dilution factor, against human and mouse DCC showed reactivity towards human and mouse DCC with observed EC₅₀ values of 0.72 nM and 1 nM for human and mouse DCC, respectively.

The flow histogram shown in this report is using one primary antibody concentration (red; 15 µg/mL) from a larger 14-concentration titration experiment (not shown; 14-point, 2.5X serial dilution from 660 nM to 4.42 pM). The isotype control (grey) was tested at a single concentration (16 µg/mL), corresponding to the third highest point in the antibody titration. Overlap between the isotype and tested antibody in the histogram reflects this difference in setup, as well as the low expression level of human DCC in CHO cells, rather than nonspecific binding.

The antibody shows clear, concentration-dependent binding to DCC despite the low target expression of human DCC.

Protocol: ExpiCHO cells were grown per manufacturer’s instruction. Plasmids expressing mouse or human DCC were transiently transfected into cells together with a plasmid expressing blue fluorescent protein (BFP). Control cells were transfected with plasmid expressing full-length human B7H3 together with the BFP plasmid. Two days after transfection, cells at a density of 3 x 10⁶ cells/mL were labeled with primary antibodies.

• Primary antibody: 15 µg/mL in 1 mL Dulbecco's phosphate-buffered saline (DPBS), 0.5% BSA, with 0.9 mM Ca²⁺, 0.5 mM Mg²⁺, kept at 4 °C. 50 µL of antibody added to cells and incubated on ice for 30 min.
• Washing conditions: Cells pelleted and washed twice in 0.5% DPBS, 0.5% BSA, with 0.9 mM Ca²⁺, 0.5 mM Mg²⁺.
• Secondary antibody: 2 µg/mL Alexa Fluor 647 F(ab')₂ goat anti-rabbit IgG Fc fragment (Jackson ImmunoResearch, 111-606-046) in 0.5% DPBS, 0.5% BSA, with 0.9 mM Ca²⁺, 0.5 mM Mg²⁺, 30 min, on ice.
• Detection: Labeled cells were analyzed with an Intellicyt iQue Screener Plus flow cytometer. Cells were first gated for positive BFP expression and then gated for the Alexa 647-conjugated secondary antibody.