ACT8 Antibody

Experimental Design for Using ACT8 Antibody in Flow Cytometry

• Question: How should I design an experiment to use the ACT8 antibody in flow cytometry for identifying CD103-expressing cells?

• Answer:

  • Preparation: Ensure that your cell samples are properly prepared for flow cytometry. This includes isolating the cells of interest (e.g., intraepithelial lymphocytes) and staining them with the ACT8 antibody conjugated to a suitable fluorochrome (e.g., PerCP-eFluor 710) .

  • Instrument Setup: Use a flow cytometer capable of detecting the chosen fluorochrome. For PerCP-eFluor 710, set up the instrument to excite at 488 nm and detect emission at 710 nm .

  • Data Analysis: Use appropriate gating strategies to identify CD103-positive cells. This may involve excluding dead cells and debris, followed by gating on the specific cell population of interest based on forward and side scatter properties.

Data Interpretation and Contradiction Analysis

• Question: How do I interpret and reconcile discrepancies in data when using ACT8 antibody for staining intraepithelial lymphocytes versus peripheral blood lymphocytes?

• Answer:

  • Interpretation: ACT8 antibody primarily targets CD103, which is highly expressed on intraepithelial lymphocytes but less so on peripheral blood lymphocytes . Discrepancies might arise from differences in cell preparation, antibody concentration, or staining conditions.

  • Contradiction Analysis:

    Variable	Intraepithelial Lymphocytes	Peripheral Blood Lymphocytes
    CD103 Expression	High	Low
    ACT8 Reactivity	Strong	Weak

    • Consider factors like cell activation status and tissue origin when interpreting results. In vitro activation can upregulate CD103 expression, potentially affecting staining intensity .

Advanced Research Questions: Molecular Target and Epitope Specificity

• Question: What is the molecular structure recognized by the ACT8 antibody, and how does it relate to its specificity for CD103?

• Answer:

  • Molecular Structure: The ACT8 antibody targets a trimeric molecule with components of 150, 125, and 105 kilodaltons, with the epitope localized on the 150 kilodalton chain. This chain contains an O-linked carbohydrate moiety .

  • Specificity: The specificity of ACT8 for CD103 (integrin alpha E) is crucial for its use in identifying mucosa-associated lymphocytes. Cross-blocking studies indicate that ACT8 sees different epitopes than other antibodies like B-Ly7 .

Methodological Considerations for Tissue Staining

• Question: How should I optimize immunohistological staining protocols using ACT8 antibody for tissue sections?

• Answer:

  • Tissue Preparation: Ensure proper fixation and sectioning of tissues. Paraffin-embedded sections are commonly used for immunohistochemistry.

  • Staining Protocol: Use a standard immunohistochemistry protocol with appropriate blocking steps to reduce non-specific binding. The ACT8 antibody can be used to stain intraepithelial lymphocytes in gut tissues, among other mucosal sites .

  • Controls: Include negative controls (e.g., omission of primary antibody) and positive controls (e.g., known CD103-expressing tissues) to validate staining specificity.

Advanced Applications in Disease Diagnosis

• Question: How can the ACT8 antibody be utilized in the diagnosis of specific lymphomas or leukemias?

• Answer:

  • Diagnostic Utility: ACT8 is useful for diagnosing gut T cell-derived T cell lymphomas and other extranodal T cell lymphomas, as well as hairy cell leukemia, due to its selective reactivity with CD103-expressing cells .

  • Clinical Application: In clinical settings, ACT8 can help differentiate between various types of lymphomas based on CD103 expression patterns. This is particularly valuable for identifying T cell lymphomas associated with coeliac disease .