aipoch/flow-cytometry-gating-strategist

Skill: Flow Cytometry Gating Strategist

Recommend optimal flow cytometry gating strategies for given cell types and fluorophores.

Basic Information

• ID: 103
• Name: Flow Cytometry Gating Strategist
• Purpose: Flow cytometry data analysis and gating strategy recommendations

Usage

Command Line

# Recommended format: comma-separated cell types and fluorophores
python scripts/main.py "CD4+ T cells,CD8+ T cells" "FITC,PE,APC"

# Or specify parameters separately
python scripts/main.py --cell-types "CD4+ T cells,CD8+ T cells" --fluorophores "FITC,PE,APC"

# Support more options
python scripts/main.py \
  --cell-types "B cells" \
  --fluorophores "FITC,PE,PerCP-Cy5.5,APC" \
  --instrument "BD FACSCanto II" \
  --purpose "cell sorting"

Parameters

| Parameter | Type | Default | Required | Description | |-----------|------|---------|----------|-------------| | --cell-types | string | - | Yes | Comma-separated list of cell types (e.g., "CD4+ T cells,CD8+ T cells") | | --fluorophores | string | - | Yes | Comma-separated list of fluorophores (e.g., "FITC,PE,APC") | | --instrument | string | - | No | Flow cytometer model (e.g., "BD FACSCanto II") | | --purpose | string | analysis | No | Purpose (analysis, cell sorting, screening) | | --output, -o | string | stdout | No | Output file path for JSON results |

Output Format

{
  "recommended_strategy": {
    "name": "Sequential Gating Strategy",
    "description": "Gating based on FSC-A/SSC-A, followed by fluorescence intensity analysis",
    "steps": [
      {
        "step": 1,
        "gate": "FSC-A vs SSC-A",
        "purpose": "Identify target cell population, exclude debris and dead cells",
        "recommendation": "Set oval gate in lymphocyte region"
      }
    ]
  },
  "fluorophore_recommendations": [
    {
      "fluorophore": "FITC",
      "channel": "BL1",
      "detector": "530/30",
      "considerations": ["May spillover with GFP"]
    }
  ],
  "panel_optimization": {
    "suggestions": ["Recommend pairing weakly expressed antigens with bright fluorophores"],
    "avoid_combinations": ["FITC and GFP used simultaneously"]
  },
  "compensation_notes": ["FITC and PE require careful compensation"],
  "quality_control": ["Recommend setting FMO controls", "Use viability dyes to exclude dead cells"]
}

Supported Cell Types

• T cells: CD4+ T cells, CD8+ T cells, Treg cells, Th1, Th2, Th17, γδ T cells
• B cells: B cells, Plasma cells, Memory B cells, Naive B cells
• Myeloid cells: Monocytes, Macrophages, Dendritic cells, Neutrophils, Eosinophils
• Stem cells: HSC, MSC, iPSC
• Tumor cells: Tumor cells, Cancer stem cells
• Others: NK cells, NKT cells, Platelets, Erythrocytes

Supported Fluorophores

| Fluorophore | Excitation Wavelength | Emission Wavelength | Detection Channel | |------|---------|---------|---------| | FITC | 488nm | 525nm | BL1 | | PE | 488nm | 575nm | YL1/BL2 | | PerCP | 488nm | 675nm | RL1 | | PerCP-Cy5.5 | 488nm | 695nm | RL1 | | PE-Cy7 | 488nm | 785nm | RL2 | | APC | 640nm | 660nm | RL1 | | APC-Cy7 | 640nm | 785nm | RL2 | | BV421 | 405nm | 421nm | VL1 | | BV510 | 405nm | 510nm | VL2 | | BV605 | 405nm | 605nm | VL3 | | BV650 | 405nm | 650nm | VL4 | | BV785 | 405nm | 785nm | VL6 | | DAPI | 355nm | 461nm | UV | | PI | 488nm | 617nm | YL2 |

Gating Strategy Types

1. Sequential Gating

Applicable scenario: Simple immunophenotyping analysis

• FSC-A/SSC-A → Exclude debris/dead cells → Fluorescence intensity analysis

2. Boolean Gating

Applicable scenario: Complex cell subset analysis

• Use logical operators (AND, OR, NOT) to define cell populations

3. Dimensionality Reduction Gating

Applicable scenario: High-dimensional data (>15 colors)

• t-SNE/UMAP visualization-assisted gating

4. Unsupervised Clustering

Applicable scenario: Discovery of unknown cell populations

• FlowSOM, PhenoGraph and other algorithms

Notes

1. Spectral Overlap Compensation: Multi-color panels must undergo compensation calculation
2. Control Setup: Must use FMO (fluorescence minus one) and isotype controls
3. Dead Cell Exclusion: Strongly recommend using viability dyes
4. Instrument Calibration: Perform QC and standard bead detection before experiments

Dependencies

• Python 3.8+
• No external dependencies (pure Python standard library)

Version

v1.0.0 - Initial version, supports basic gating strategy recommendations

Risk Assessment

| Risk Indicator | Assessment | Level | |----------------|------------|-------| | Code Execution | Python scripts with tools | High | | Network Access | External API calls | High | | File System Access | Read/write data | Medium | | Instruction Tampering | Standard prompt guidelines | Low | | Data Exposure | Data handled securely | Medium |

Security Checklist

• [ ] No hardcoded credentials or API keys
• [ ] No unauthorized file system access (../)
• [ ] Output does not expose sensitive information
• [ ] Prompt injection protections in place
• [ ] API requests use HTTPS only
• [ ] Input validated against allowed patterns
• [ ] API timeout and retry mechanisms implemented
• [ ] Output directory restricted to workspace
• [ ] Script execution in sandboxed environment
• [ ] Error messages sanitized (no internal paths exposed)
• [ ] Dependencies audited
• [ ] No exposure of internal service architecture

Prerequisites

No additional Python packages required.

Evaluation Criteria

Success Metrics

• [ ] Successfully executes main functionality
• [ ] Output meets quality standards
• [ ] Handles edge cases gracefully
• [ ] Performance is acceptable

Test Cases

1. Basic Functionality: Standard input → Expected output
2. Edge Case: Invalid input → Graceful error handling
3. Performance: Large dataset → Acceptable processing time

Lifecycle Status

• Current Stage: Draft
• Next Review Date: 2026-03-06
• Known Issues: None
• Planned Improvements:
  • Performance optimization
  • Additional feature support