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Flow Cytometry for Anti-Glypican 4 (GPC4) [IPI-mGPC4.9]

Addgene #222516

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 22, 2024
Publication Date: October 23, 2024 (modified November 11, 2024)
DOI: https://doi.org/10.57733/addgene.iysjin
Abstract: Experimental results for Anti-glypican 4 (GPC4) [IPI-mGPC4.9] (Addgene #222516) in a flow cytometry assay with a human sample. Anti-glypican 4 (GPC4) [IPI-mGPC4.9] is specific for human and mouse GPC4 and weakly cross-reactive with mouse GPC6.
Citation: Flow Cytometry for Anti-Glypican 4 (GPC4) [IPI-mGPC4.9]. Anderson Z, Zhu H, Riedel T, Li H, Meijers R, Moshinsky D 2024. Addgene Report, https://doi.org/10.57733/addgene.iysjin

**Caption:** Histogram overlays from FACS analysis on ExpiCHO cells transfected with human glypicans (GPC1-GPC6). Cells expressing GPCs were labeled with Anti-Glypican 4 (GPC4) [IPI-mGPC4.9] (Addgene #222516) 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 4 (GPC4) [IPI-mGPC4.9] is specific for human and mouse GPC4 (violet lines with fill) and is weakly cross-reactive with mouse GPC6 (blue line with fill). 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 4 (GPC4) [IPI-mGPC4.9]
Addgene #222516; Alexa Fluor 647 F(ab')₂ Goat Anti-Rabbit IgG Fc Fragment
Jackson ImmunoResearch #111-606-046

Materials & Methods

Sample

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

Buffer

Name: PBS

Primary Antibody

Name: Anti-Glypican 4 (GPC4) [IPI-mGPC4.9]
Source: Addgene
Catalog Number: 222516
Host Species: Rabbit
Target Antigen: GPC4
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
Host 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 GPC4; Weak cross-reactivity with mouse GPC6.