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Objectives Anti-dsDNA antibodies play a crucial role in the pathogenesis of SLE. Therefore, detailed analysis of dsDNA-specific B cells and plasma cells is of great importance but is complicated by the stickiness of dsDNA. We investigated the suitability of double-stranded DNA oligonucleotides (dsDNA oligo) for the characterization of dsDNA-specific plasma cells and B cells using flow cytometry.Methods dsDNA oligo with different lengths between 9 and 140 base pairs (bp) were synthesized by Integrated DNA Technologies (IDT). The inhibition of anti-dsDNA antibodies from serum samples of SLE patients and NZB/W mice by dsDNA oligo with lengths between 9 and 120bp was investigated using an IgG anti-dsDNA ELISA. The staining of dsDNA-specific B cells and plasma cells was performed by flow cytometry using a 100 bp dsDNA oligo labeled with 6-FAM and Cy5. The intracellular flow cytometric staining of plasma cells was compared with the ELISPOT results.Results The reactivity of dsDNA oligos with anti-dsDNA antibodies is length-dependent and sequence-independent. dsDNA oligos longer than 80 bp react with most anti-dsDNA antibodies. The 100 bp dsDNA oligo achieved approximately 85% inhibition of anti-dsDNA antibody binding to dsDNA at a concentration of 500 nM in an ELISA. Four 100 bp dsDNA oligos with different nucleotide sequences showed comparable blocking patterns. Next, we investigated the detection of dsDNA-specific plasma cells in NZB/W mice and SLE patients. In spleen cells and bone marrow of NZB/W mice, double-positive intracellular staining was observed within the plasma cell population (CD138+ intracellular kappa+). A dsDNA-specific plasma cell staining (CD19+ CD27++) was also observed in PBMCs from active SLE patients. Pre-blocking experiments with unconjugated 100-bp dsDNA oligo confirmed the specificity of the detection. Comparable results were obtained in ELISPOT using calf thymus DNA. In addition, we tested the suitability of the 100 bp dsDNA oligo for surface double staining to detect dsDNA-specific B cells. Using this approach, dsDNA-specific B cell populations were detected in samples from both NZB/W mice and SLE patients.Conclusions The use of a 100 bp dsDNA oligo can be a valuable tool for the in-depth analysis of autoreactive dsDNA-specific B cells and plasma cells, even at the single-cell level.