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
- December 13, 2024
- Publication Date
- December 17, 2024 (modified December 19, 2024)
- DOI
- https://doi.org/10.57733/addgene.ihsx47
- Abstract
- Experimental results for Anti-Glypican 4 (GPC4) [IPI-mGPC4.9] (Addgene #222516) in a flow cytometry assay with ExpiCHO cells expressing human GPC4.
- Citation
-
Flow Cytometry for Anti-Glypican 4 (GPC4) [IPI-mGPC4.9]. Anderson Z, Li H, Riedel T, Zhu H, Meijers R, Moshinsky D 2024. Addgene Report, https://doi.org/10.57733/addgene.ihsx47
![Anti-Glypican 4 (GPC4) [IPI-mGPC4.9] (Addgene #222516) recognizes mouse and human GPC4. Histogram from FACS analysis on ExpiCHO cells transfected with mouse or human GPC4 (red), or B7H3 negative control (gray). Cells expressing mouse (left panel) or human (right panel) GPC4 were labeled with Anti-Glypican 4 (GPC4) [IPI-mGPC4.9] and Alexa Fluor 647 F(ab’)2 goat anti-rabbit IgG Fc fragment (Jackson ImmunoResearch, 111-606-046). Labeled cells were analyzed with an Intellicyt iQue Screener Plus flow cytometer. Histograms were generated and normalized to mode using FlowJo™ v10.10.](https://datahub-media.addgene.org/report-images/ihsx47/GPC_CellDisplay_4.9.png)
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
- Species
- Human
- Cell / Tissue Type
- ExpiCHO
- Additional Information
- ExpiCHO cells were transiently transfected with human GPC4 or B7H3 (negative control).
Buffer
- Name
- PBS
Primary Antibody
- Name
- Anti-Glypican 4 (GPC4) [IPI-mGPC4.9]
- Source
- Addgene
- Catalog Number
- 222516
- Species
- Rabbit
- Gene / Target
- GPC4
- Concentration
- 5 µg/mL
- Time
- 30 min
- Temperature
- On ice
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. Plasmids expressing mouse or human GPC4 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: 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
- Recognizes mouse and human GPC4