Abstract
We have developed a series of monovalent fluorophore-conjugated affinity probes based on the hapten 3-nitro-4-hydroxy-5-iodophenylacetyl (NIP), which is widely used as a model antigen to study B lymphocytes and the functional principles of B cell antigen receptors (BCRs). We successfully used them in flow-cytometry, confocal and super-resolution microscopy techniques.
Highlights
Selda Kabatas Glowacki,‡a,b Maria Angela Gomes de Castro, ‡a Ka Man Yip,c Ommolbanin Asadpour,a,b Matthias Münchhalfen,d Niklas Engels*d and Felipe Opazo *a,b
We have developed a series of monovalent fluorophore-conjugated affinity probes based on the hapten 3-nitro-4-hydroxy-5iodophenylacetyl (NIP), which is widely used as a model antigen to study B lymphocytes and the functional principles of B cell antigen receptors (BCRs)
We successfully used them in flow-cytometry, confocal and super-resolution microscopy techniques
Summary
We developed a series of fluorescent monovalent affinity probes for NIP-reactive BCRs that proved suitable for flow cytometry and Stimulated Emission-Depletion (STED) super-resolution microscopy (Fig. 1) To generate these NIP-based affinity probes, we chose different peptide spacers with good solubility in water yet varying net charge. We observed minimal differences in their emission intensities, with KM7 being a little less bright than the pure fluorophore Star635P These measurements suggest that it is not the photophysical properties of the probes that are decisive for the differences in cellular fluorescence intensity, but rather the binding of the probes to the NIP-reactive BCRs. we stained Ramos IgDNIP cells with various concentrations of the 5 different NIP-probes and imaged them live or after chemical fixation with 4% paraformaldehyde (ESI Fig. S3 and S4†). KM1, KM7 and KM8 were designed with internal lysines (K) to enhance retention after fixation with
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