X-linked agammaglobulinaemia (XLA) is an immune deficiency characterised by absence of mature B cells due to mutations in the Bruton’s tyrosine kinase (Btk) gene. A previous study demonstrated Btk gene expression in an XLA mouse model reconstituted the ability of the haematopoietic compartment to produce mature B cells¹. This provides a rationale for treating XLA patients with gene therapy, and the impetus to proceed with necessary preclinical studies including evaluating B cell function in cell lines transduced with Btk gene.

Calcium (Ca²⁺) plays an important role in B cell activation and its signals control proliferation, differentiation, apoptosis and multiple Ca²⁺-sensitive transcription regulators. A downstream consequence of B cell receptor signaling using IgM is positive Ca²⁺ flux.

Ca²⁺ flux measures the capacity of stimulated cells to increase intracellular calcium. We investigated whether cells transduced with lentiviral vectors encoding Btk were able to respond to an IgM stimulus. The read-out from the traditional flux ratio may simply be binary (yes/no); however, in other cases quantitating the degree of flux may be more appropriate. We assessed flow cytometry-based Ca²⁺ flux assays in order to understand their limitations in providing reproducible measurements. These limitations can be observed when, i) limiting cell numbers per sample are present, which is especially pertinent to mouse specimens, ii) repeating Ca²⁺ flux measurements over time and iii) examining different cell lines. We demonstrate that measuring the degree of Ca²⁺ flux using dot plots, rather than the traditional flux ratios, may be preferable when limited cells are available for analysis.