Apr 24, 2026
Flow Cytometry Protocol for Human HER2 (ERBB2/CD340) Detection Using Research-Grade Anti-HER2 Antibodies
- abinScience cheng1
- 1abinScience, AtaGenix Laboratories, Wuhan, China
External link: https://www.abinscience.com
Protocol Citation: abinScience cheng 2026. Flow Cytometry Protocol for Human HER2 (ERBB2/CD340) Detection Using Research-Grade Anti-HER2 Antibodies. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lyormmgx9/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 24, 2026
Last Modified: April 24, 2026
Protocol Integer ID: 315647
Keywords: HER2, ERBB2, CD340, flow cytometry, breast cancer, receptor tyrosine kinase, surface staining, flow cytometry protocol for human her2, human her2 monoclonal, detecting her2, flow cytometry protocol, human her2, her2, tumor sample, expression on human cell, applicable to cultured cell line, cultured cell line, erbb2, human cell, cell
Disclaimer
For research use only. Not for diagnostic or therapeutic purposes.
Abstract
This protocol describes a surface staining procedure for detecting HER2 (ERBB2/CD340) expression on human cells by flow cytometry. HER2 is a receptor tyrosine kinase frequently overexpressed in breast, gastric, and other solid tumors. This protocol has been optimized for use with abinScience research-grade anti-human HER2 monoclonal antibodies, including directly conjugated formats (FITC, PE, APC), and is applicable to cultured cell lines and dissociated tumor samples.
Cell Preparation
1. Harvest cells. For adherent cells (e.g., SK-BR-3, BT-474), use Accutase to preserve surface antigens.
2. Wash once with PBS, centrifuge at 300 × g for 5 minutes.
3. Resuspend in FACS buffer (PBS + 2% FBS + 0.05% sodium azide) at 1 × 10⁷ cells/mL.
4. Aliquot 100 μL (1 × 10⁶ cells) per well into a 96-well V-bottom plate.
Recommended positive control: SK-BR-3 (HER2 3+) or BT-474 (HER2 3+).
Recommended negative control: MDA-MB-231 (HER2 low).
Viability Staining
1. Centrifuge at 300 × g for 5 minutes, discard supernatant.
2. Resuspend in 100 μL PBS containing viability dye per manufacturer's instructions.
3. Incubate at room temperature for 15 minutes, protected from light.
4. Wash once with FACS buffer.
Fc Blocking
1. Resuspend cells in 50 μL FACS buffer containing Fc block (5 μL per test).
2. Incubate at room temperature for 10 minutes.
3. Do NOT wash. Proceed directly to antibody staining.
Primary Antibody Staining
Option A — Directly conjugated antibodies (recommended for simplicity):
- Anti-Human HER2 Antibody (4D5V8), FITC (Cat# HY286117): https://www.abinscience.com/product/40/4706.html
- Anti-Human HER2 Antibody (4D5V8), PE (Cat# HY286127): https://www.abinscience.com/product/40/7727.html
- Anti-Human HER2 Antibody (4D5V8), APC (Cat# HY286137): https://www.abinscience.com/product/40/5713.html
Option B — Unconjugated antibodies (requires secondary staining):
- Anti-Human HER2 Antibody (4D5V8) (Cat# HY286107): https://www.abinscience.com/product/40/3823.html
- Anti-Human HER2 Antibody (MAB 2C4) (Cat# HY286207): https://www.abinscience.com/product/40/3824.html
- Research Grade Trastuzumab (Cat# HY286016): https://www.abinscience.com/product/71/2222.html
1. Add antibody at recommended concentration: 1–10 μg/mL for unconjugated; per manufacturer's datasheet for conjugated formats.
2. Incubate at 4°C for 30 minutes, protected from light.
3. Wash twice with 200 μL FACS buffer, centrifuge at 300 × g for 5 minutes.
Secondary Antibody Staining (Option B only)
1. Resuspend in 100 μL FACS buffer containing fluorochrome-conjugated secondary antibody at recommended dilution.
2. Incubate at 4°C for 20–30 minutes, protected from light.
3. Wash twice with FACS buffer.
Fixation (Optional)
1. Resuspend cells in 200 μL 2% paraformaldehyde in PBS.
2. Incubate at 4°C for 15 minutes.
3. Wash once with FACS buffer.
4. Resuspend in 200 μL FACS buffer for acquisition.
Fixed samples can be stored at 4°C protected from light and acquired within 24 hours.
Acquisition and Analysis
1. Acquire ≥10,000 events in the live cell gate on a flow cytometer.
2. Gating strategy: FSC/SSC → singlets (FSC-A vs FSC-H) → viable cells → HER2+ population.
3. Include isotype control or FMO control to set the positive gate.
4. Expected results: SK-BR-3 should show >95% HER2-positive; MDA-MB-231 should show <5% HER2-positive.