Flow Cytometry for Anti-SEMA3C [IPI-mSEM3C.5]
Addgene #254647
- Data Submitted By
- Zachary Anderson , Haiying Li , Travis Riedel , Haisun Zhu and Deborah Moshinsky
- Lab Name
- Institute for Protein Innovation (IPI)
- Submission Date
- April 16, 2026
- Publication Date
- May 12, 2026 (modified May 14, 2026)
- DOI
- https://doi.org/10.57733/addgene.6esgg5
- Abstract
- Experimental results for Anti-SEMA3C [IPI-mSEM3C.5] (Addgene #254647) in a flow cytometry assay with ExpiCHO cells expressing human or mouse SEMA3C.
- Citation
-
Flow Cytometry for Anti-SEMA3C [IPI-mSEM3C.5]. Anderson Z, Li H, Riedel T, Zhu H, Moshinsky D 2026. Addgene Report, https://doi.org/10.57733/addgene.6esgg5
![Anti-SEMA3C [IPI-mSEM3C.5] (Addgene #254647) recognizes human and mouse SEMA3C. Histogram from FACS analysis on ExpiCHO cells transfected with human or mouse SEMA3C (red), or B7H3 negative control (gray). Cells expressing human (left panel) or mouse (right panel) SEMA3C were labeled with Anti-SEMA3C [IPI-mSEM3C.5] 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/6esgg5/SEMA3C.5_Flow.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-SEMA3C [IPI-mSEM3C.5]
Addgene #254647 -
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 cells
- Additional Information
- ExpiCHO cells were transiently transfected with human SEMA3C or B7H3 (negative control).
Buffer
- Name
- PBS
Primary Antibody
- Name
- Anti-SEMA3C [IPI-mSEM3C.5]
- Source
- Addgene
- Catalog Number
- 254647
- Host Species
- Rabbit
- Target Antigen
- SEMA3C
- Concentration
- 15 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
- Concentration
- 2 µg/mL
- Time
- 30 min
- Temperature
- On ice
Additional Information
- Additional Information
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 SEMA3C showed reactivity towards human and mouse SEMA3C with observed EC50 values of 38.93 nM and 31.46 nM for human and mouse SEMA3C, respectively.
Protocol: ExpiCHO cells were grown per manufacturer’s instruction. Plasmids expressing mouse or human SEMA3C 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.
- Controls
- Control cells were transfected with plasmid expressing full-length human B7H3 together with the BFP plasmid.
Results
- Result
- Pass (The antibody worked under these conditions)
- Describe Results
- Recognizes human and mouse SEMA3C