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Updated: Aug 9, 2026
Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry
Published on: March 28, 2014
Calibration of fluorescence intensities to quantify antibody binding surface determinants of cell subpopulations by
R Dux1, A Kindler-Röhrborn, K Lennartz
1Institute of Cell Biology (Cancer Research), West German Cancer Center Essen.
Insights
This study quantifies antibody binding sites on rat brain cells using flow cytometry. Researchers determined cell proportions and binding site numbers during development.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Cell surface differentiation antigens play crucial roles in neural development.
- Monoclonal antibody (Mab) RB13-6 recognizes a specific antigen on rat brain cells.
- Understanding antigen expression levels is key to studying cell subpopulations.
Purpose of the Study:
- To quantitatively determine the mean number of antibody binding sites per cell.
- To analyze antigen expression in a low-level expressing subpopulation of rat brain cells.
- To assess antigen-positive cell proportions and binding site density during rat brain development.
Main Methods:
- Quantitative indirect immunofluorescence analysis via flow cytometry.
- Statistical methods to define cut-off borders for antigen-positive/negative cells.
- Flow cytometer calibration using a clonal cell line with pre-determined binding site numbers (radioimmunoassay, Scatchard-plot analysis).
Main Results:
- Developed a method to quantify antibody binding sites on low-expressing antigen cells.
- Successfully determined the proportion of Mab RB13-6 binding brain cells.
- Quantified the mean number of Mab RB13-6 binding sites per binding cell as a function of developmental stage.
Conclusions:
- The analytical procedure allows for precise quantification of cell surface antigen expression.
- This method is valuable for studying antigen dynamics in specific cell subpopulations during development.
- Provides insights into the developmental regulation of cell surface molecules in the rat brain.
Abstract:
Quantitative indirect immunofluorescence analysis by flow cytometry was used to determine the mean number of antibody binding sites per cell in a small subpopulation of rat brain cells expressing low levels of a cell surface differentiation antigen recognized by monoclonal antibody (Mab) RB13-6 (Kindler-Röhrborn et al.: Differentiation 30:53-60, 1985). For these non-disjunct distributions of fluorescence intensities, the cut-off border between antigen-positive and antigen-negative cells was defined by a statistical test. To eliminate the influence of accidental disturbances leading to incorrect statistical decisions, the curves for antigen-negative cells were fitted according to cell number and shape. The flow cytometer was calibrated with the use of a clonal cell line for which the average number of Mab RB13-6 binding sites per cell had previously been determined by radioimmunoassay and Scatchard-plot analysis. Using this analytical procedure, both the proportion of Mab binding brain cells and the mean number of Mab binding sites per Mab binding cell could be determined as a function of developmental stage.
