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05
Jan
How to Use Anti-Human CD3 Antibody in Flow Cytometry (FACS)
Practical CD3 FACS Staining Guide for Human T Cell Analysis
Flow cytometry is one of the most widely used techniques for analyzing immune cell populations. Among all T cell markers, CD3 is considered the most reliable pan-T cell marker for human samples.
This guide explains how to use anti-human CD3 antibodies in flow cytometry (FACS), covering antibody selection, staining workflow, controls, and data interpretation for accurate human CD3 flow cytometry.
Why CD3 Is Used in Flow Cytometry
CD3 is a multi-subunit protein complex associated with the T cell receptor (TCR). Because it is expressed on nearly all mature T cells, CD3 is routinely used as a foundational marker in FACS-based immunophenotyping.
- 🔬 Pan-marker for total human T cells
- 📊 Compatible with CD4/CD8 subset analysis
- ⚙️ Suitable for fresh or cryopreserved PBMCs
Choosing the Right CD3 Flow Cytometry Antibody
Selecting an appropriate CD3 flow cytometry antibody is essential for obtaining clean and reproducible data. Key factors include antibody clone, fluorophore, and species reactivity.
| Selection Factor | Recommendation |
|---|---|
| Clone | Well-validated monoclonal clones for human CD3 |
| Fluorophore | Match panel design and instrument configuration |
| Application Validation | Flow cytometry (FACS) tested |
Step-by-Step CD3 FACS Staining Protocol
- Prepare single-cell suspension (e.g., PBMCs) and wash with FACS buffer.
- Block Fc receptors if required to reduce nonspecific binding.
- Add fluorophore-conjugated anti-human CD3 antibody at recommended dilution.
- Incubate for 20–30 minutes at 4°C in the dark.
- Wash cells thoroughly and resuspend for acquisition.
- Acquire data on a calibrated flow cytometer.
Controls and Data Interpretation
Proper controls are essential for accurate CD3 FACS analysis. Include unstained cells, single-stained controls, and fluorescence minus one (FMO) controls when designing multicolor panels.
CD3-positive populations should form a clearly separated cluster, enabling reliable gating of total T cells prior to downstream subset analysis.
Common Troubleshooting Tips
- High background: optimize antibody dilution and washing steps.
- Weak signal: verify antibody clone and fluorophore brightness.
- Poor resolution: review panel design and compensation settings.
Biofargo team
