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Flow Cytometry for Anti-Netrin-1 [IPI-NTN1.33]

Addgene #241893

Data Submitted By: Zachary Anderson , Haiying Li , Travis Riedel , Haisun Zhu and Deborah Moshinsky
Lab Name: Institute for Protein Innovation (IPI)
Submission Date: November 24, 2025
Publication Date: November 26, 2025 (modified December 17, 2025)
DOI: https://doi.org/10.57733/addgene.v5fzxa
Abstract: Experimental results for Anti-Netrin-1 [IPI-NTN1.33] (Addgene #241893) in a flow cytometry assay with ExpiCHO cells expressing human or mouse NTN1.
Citation: Flow Cytometry for Anti-Netrin-1 [IPI-NTN1.33]. Anderson Z, Li H, Riedel T, Zhu H, Moshinsky D 2025. Addgene Report, https://doi.org/10.57733/addgene.v5fzxa

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

Antibodies Used: Anti-Netrin-1 [IPI-NTN1.33]
Addgene #241893; Alexa Fluor 647 F(ab')2 goat anti-rabbit IgG, Fc fragment specific
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 NTN1 or B7H3 (negative control).

Buffer

Name: PBS

Primary Antibody

Name: Anti-Netrin-1 [IPI-NTN1.33]
Source: Addgene
Catalog Number: 241893
Host Species: Rabbit
Target Antigen: NTN1
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 specific
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 NTN1 showed reactivity towards human and mouse NTN1 with observed EC₅₀ values of 10.12 nM and 7.99 nM for human and mouse NTN1, respectively.

Protocol: ExpiCHO cells were grown per manufacturer’s instruction. Plasmids expressing mouse or human NTN1 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 Netrin-1