Flow Cytometry for Anti-Glypican 1 (GPC1) [IPI-GPC1.44]

Protocol: ExpiCHO cells were grown per manufacturer’s instruction. Human and mouse GPC1-GPC6 were cloned in separate expression plasmids and 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 2 x 10⁶ cells/mL were labeled with primary antibodies.

Primary antibody: 5 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.