Two‐Color Analysis of Leukocytes Labeled by Modified RBCs and their Fragments

Red blood cells (RBCs) are attractive tools for surface modification to adhere specifically to molecules, cellular fragments (e.g., microvesicles), or whole cells for potential use in bioanalytical assays or as a delivery vehicle in targeted therapy. Within this study, we have loaded RBCs with fluorochrome‐conjugated antibodies (Ab) against CD45 and CD22 leukocyte markers and evaluated the conjugation process by microscopy. We have assessed the potential application of RBCs fragments generated from conjugated RBCs for targeting Cyto‐Trol control cells by flow cytometric (FCM) approaches. Based on their scattering and fluorescence characteristics (FITC and PE expression), modified RBCs and their fragments, Cyto‐Trol cells, and clusters of both were distinguished by two color FCM analysis. Fragments with anti‐human Kallestad Ab as a nonspecific FITC conjugate had less than 20% binding to Cyto‐Trol controls compared to CD45‐FITC Ab conjugate with nearly 100% binding capacity. Cyto‐Trol‐microvesicle‐clusters were more than 45% positive for either FITC or PE. Anti‐CD22‐PE modified RBCs fragments were also useful in staining and showing about 19.5% positively stained events in the Cyto‐Trol region. The proof‐of‐concept shows, that specific antibody can be attached to RBCs, and generated fragments can be useful to stain target cells for FCM analysis.