Return to Antibody Results

Flow Cytometry for Anti-SEMA3A [IPI-SEM3A.17]

Addgene #254646

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.c089j2
Abstract: Experimental results for Anti-SEMA3A [IPI-SEM3A.17] (Addgene #254646) in a flow cytometry assay with ExpiCHO cells expressing human or mouse SEMA3A.
Citation: Flow Cytometry for Anti-SEMA3A [IPI-SEM3A.17]. Anderson Z, Li H, Riedel T, Zhu H, Moshinsky D 2026. Addgene Report, https://doi.org/10.57733/addgene.c089j2

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

Antibodies Used: Anti-SEMA3A [IPI-SEM3A.17]
Addgene #254646; 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 SEMA3A or B7H3 (negative control).

Buffer

Name: PBS

Primary Antibody

Name: Anti-SEMA3A [IPI-SEM3A.17]
Source: Addgene
Catalog Number: 254646
Host Species: Rabbit
Target Antigen: SEMA3A
Concentration: 15 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
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 SEMA3A showed reactivity towards human and mouse SEMA3A with observed EC₅₀ values of 0.54 nM and 0.54 nM for human and mouse SEMA3A, respectively.

Protocol: ExpiCHO cells were grown per manufacturer’s instruction. Plasmids expressing mouse or human SEMA3A 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.

Protocol

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 SEMA3A