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Flow Cytometry for Anti-Glypican 3 (GPC3) [IPI-mGPC3.19]

Addgene #222515

Data Submitted By: Zachary Anderson , Haiying Li , Travis Riedel , Haisun Zhu , Rob Meijers and Deborah Moshinsky
Lab Name: Institute for Protein Innovation (IPI)
Submission Date: October 29, 2024
Publication Date: October 30, 2024 (modified November 11, 2024)
DOI: https://doi.org/10.57733/addgene.16x4s7
Abstract: Experimental results for Anti-glypican 3 (GPC3) [IPI-mGPC3.19] (Addgene #222515) in a flow cytometry assay with a mouse sample. Anti-glypican 3 (GPC3) [IPI-mGPC3.19] is specific for human and mouse GPC3 and does not recognize other human or mouse GPCs.
Citation: Flow Cytometry for Anti-Glypican 3 (GPC3) [IPI-mGPC3.19]. Anderson Z, Li H, Riedel T, Zhu H, Meijers R, Moshinsky D 2024. Addgene Report, https://doi.org/10.57733/addgene.16x4s7

**Caption:** Histogram overlays from FACS analysis on ExpiCHO cells transfected with mouse glypicans (GPC1-GPC6). Cells expressing GPCs were labeled with Anti-Glypican 3 (GPC3) [IPI-mGPC3.19] (Addgene #222515) and Alexa Fluor 647 F(ab’)2 Fragment Goat Anti-Rabbit IgG Fc (Jackson ImmunoResearch, 111-606-046). Labeled cells were analyzed with an Intellicyt iQue Screener Plus flow cytometer. Anti-Glypican 3 (GPC3) [IPI-mGPC3.19] is specific for human and mouse GPC3 (orange lines with fill) and does not recognize other GPCs. Histograms were normalized to mode and displayed as an overlay with half offset using FlowJo™ v10.10. Bottom table summarizes GPC cross-reactivity. Abbreviations: glypican (GPC), human (Hu), mouse (Mo), strong (++), weak (+), none ( ).
**Image attribution:** Institute for Protein Innovation (IPI)

This report is made available under the Creative Commons Attribution 4.0 International License.

Antibodies Used: Anti-Glypican 3 (GPC3) [IPI-mGPC3.19]
Addgene #222515; Alexa Fluor 647 F(ab')₂ Goat Anti-Rabbit IgG Fc Fragment
Jackson ImmunoResearch #111-606-046

Materials & Methods

Sample

Species: Mouse
Cell / Tissue Type: ExpiCHO
Additional Information: ExpiCHO cells were transiently transfected with mouse GPC1-GPC6 or B7H3 (negative control).

Buffer

Name: PBS

Primary Antibody

Name: Anti-Glypican 3 (GPC3) [IPI-mGPC3.19]
Source: Addgene
Catalog Number: 222515
Species: Rabbit
Gene / Target: GPC3
Concentration: 5 µg/mL
Time: 30 min
Temperature: On ice

View in Addgene Catalog

Secondary Antibody

Name: Alexa Fluor 647 F(ab')₂ Goat Anti-Rabbit IgG Fc Fragment
Source: Jackson ImmunoResearch
Catalog Number: 111-606-046
Species: Rabbit
Concentration: 2 µg/mL
Time: 30 min
Temperature: On ice

Additional Information

Additional Information

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.

Results

Result: Pass (The antibody worked under these conditions)
Describe Results: Strong signal; Specific for human and mouse GPC3; No cross-reactivity with other GPCs.