Target

Overview of Target Identification

Definition: The target in flow cytometry refers to the specific cell type, subcellular location, or molecule that you intend to study or analyze
Importance:
- Experimental Design: The choice of target dictates the experimental design, including sample preparation, antibody selection, and staining protocols
- Data Interpretation: Understanding the characteristics of the target is essential for interpreting the data and drawing meaningful conclusions
- Assay Specificity: The target determines the specificity of the assay, ensuring that you are measuring what you intend to measure
Key Aspects of Target Identification:
- Cell Type
- Subcellular Location
- Molecule

Definition: A specific category of cells defined by their morphology, function, and surface markers
Examples:
- T cells (CD4+, CD8+)
- B cells (CD19+, CD20+)
- Macrophages (CD14+, CD68+)
- Dendritic cells (CD11c+, HLA-DR+)
- Stem cells (CD34+, CD133+)
Considerations:
- Cell Surface Markers: Identify specific cell surface markers that can be used to distinguish the target cell type from other cells in the sample
- Cell Lineage: Consider the lineage and differentiation stage of the target cell type
- Cell Function: Consider the function of the target cell type and how it may be affected by the experimental conditions
Antibody Selection:
- Choose antibodies that are specific for the cell surface markers of the target cell type
- Select antibodies that have been validated for flow cytometry
- Consider using a combination of antibodies to improve specificity
Gating Strategies:
- Develop a gating strategy to identify the target cell type based on its cell surface markers
- Use appropriate controls to validate the gating strategy
- Consider using hierarchical gating to identify subsets of the target cell type
Examples of cell type analysis:
- Blood: Characterize leukocyte populations based on the surface markers
- Tissue: Identify the cell types present in the tissue microenvironment

Definition: The specific compartment or structure within a cell where a molecule or process of interest is located
Examples:
- Nucleus
- Cytoplasm
- Mitochondria
- Endoplasmic reticulum
- Golgi apparatus
Considerations:
- Intracellular Targets: Identify the specific intracellular targets that you want to study
- Subcellular Localization: Determine the subcellular location of the target molecules
- Cell Permeabilization: Use appropriate permeabilization methods to allow antibodies to access intracellular targets
Staining Protocols:
- Use appropriate fixation and permeabilization methods to preserve cell structure and allow antibody access
- Optimize staining protocols to maximize signal intensity and minimize background noise
- Consider using fluorescent dyes that are specific for certain organelles
Imaging Flow Cytometry:
- Use imaging flow cytometry to visualize the subcellular localization of target molecules
- Use appropriate image analysis techniques to quantify the localization
Applications:
- Investigate molecular events: Track the proteins expressed by the organelle at a given time point
- Assess the changes in the proteins expression levels: Determine the cellular event

Definition: The specific molecule (e.g., protein, nucleic acid, lipid) that you want to study or analyze
Examples:
- Cytokines (IL-2, TNF-α)
- Transcription factors (NF-κB, STAT3)
- Cell cycle proteins (Cyclin D1, p53)
- Surface receptors (CD4, CD8)
Considerations:
- Expression Level: Consider the expression level of the target molecule and choose appropriate antibodies or probes
- Post-Translational Modifications: Consider any post-translational modifications (e.g., phosphorylation, glycosylation) that may affect antibody binding
- Protein-Protein Interactions: Consider any protein-protein interactions that may affect the availability of the target molecule
Antibody Selection:
- Choose antibodies that are specific for the target molecule and that have been validated for flow cytometry
- Select antibodies that recognize specific isoforms or post-translational modifications of the target molecule
- Consider using a combination of antibodies to improve specificity
Assay Optimization:
- Optimize the staining protocol to maximize signal intensity and minimize background noise
- Use appropriate controls to validate the assay
- Consider using signal amplification techniques to improve sensitivity
Application:
- Intracellular proteins: Understand what triggers the activation of protein

Cell Type: dictates how you collect and prepare your sample, select your surface marker antibodies, and design your gating strategy
Subcellular Location: determines whether you need permeabilization and influences your choice of dyes or specialized techniques like imaging flow cytometry
Molecule: guides your antibody selection, staining protocol optimization, and control design

Weak Signal:
- Possible Causes:
  - Low expression level of the target molecule
  - Poor antibody binding
  - Incorrect staining protocol
- Troubleshooting Steps:
  - Increase antibody concentration
  - Optimize staining protocol
  - Use signal amplification techniques
High Background Noise:
- Possible Causes:
  - Non-specific antibody binding
  - Autofluorescence
  - Contamination
- Troubleshooting Steps:
  - Use blocking reagents
  - Reduce autofluorescence
  - Clean samples
Unexpected Results:
- Possible Causes:
  - Incorrect antibody selection
  - Improper sample preparation
  - Instrument malfunction
- Troubleshooting Steps:
  - Verify antibody specificity
  - Review sample preparation protocols
  - Inspect instrument for malfunctions
Use of Controls:
- FMO (Fluorescence Minus One): Serves as a control when assessing the spread of fluorescence signal into other channels, and is important for setting accurate gates
- Isotype Control: Designed to have a similar level of nonspecific binding as the antibody, and helps estimate background signal

Target: The specific cell type, subcellular location, or molecule that you intend to study or analyze
Cell Type: A specific category of cells defined by their morphology, function, and surface markers
Subcellular Location: The specific compartment or structure within a cell where a molecule or process of interest is located
Molecule: The specific molecule (e.g., protein, nucleic acid, lipid) that you want to study or analyze
Cell Surface Marker: A protein or other molecule expressed on the surface of a cell that can be used to identify and distinguish it from other cells
Antibody: A protein produced by the immune system that binds specifically to a target molecule
Fluorophore: A fluorescent molecule that emits light when excited by a specific wavelength of light