Background

Basophils are evolutionarily conserved in vertebrates, despite their small numbers and short lifespan, suggesting that basophils have beneficial roles in maintaining health. However, these roles are not fully defined. The use of Cre-loxP approaches has allowed the generation of conditional mice with basophil deficiency, or deficiencies in basophil-derived mediators, which have significantly contributed to our understanding of basophil function in health and disease. For example, we recently demonstrated that mice with basophils lacking tumor necrosis factor (TNF) exhibited high morbidity and reduced ability to clear bacteria in the cecal ligation and puncture (CLP) mouse model of sepsis, highlighting a role for basophils in innate immunity against bacteria (Piliponsky et al., 2019). Moreover, we showed that basophils are recruited into the infection site, the peritoneal cavity, as early as 1 h after CLP. To rule out potential non-specific effects of the Cre-loxP approach, the ultimate proof for our findings was provided by rescuing mice with a deficiency in basophil-derived TNF. To do this, we adoptively transferred the basophil-enriched CD49b⁺ fraction obtained from the spleens of wild type mice into the peritoneal cavities of septic mice (Figure 1). Please note that this protocol does not compare the method used to other available methodologies, as it was specifically designed to assess the basophil-derived-TNF dependency for the enhancement of innate immune responses after CLP. More extensive studies than the ones described in this protocol are required to determine how long basophils live and how they distribute in the adoptive host.


Figure 1. Strategy to identify basophils among total peritoneal cells in mice that received an adoptive transfer of basophils after cecal ligation and puncture (CLP). Representative flow cytometry plots and frequencies used to identify basophils amongst peritoneal cells obtained from mice that received an adoptive transfer with the CD49b⁺ basophil-enriched fraction of splenocytes (4 x 10⁵ cells) at 3 h after induction of CLP of moderate severity (50% ligation, one puncture with a 22G needle). A and B. FcεRiα⁺, c-Kit^- (A) and CD49b⁺ (B) cells were gated and defined as basophils amongst peritoneal cells obtained from mice at 24 h after CLP.

Here, we describe such a protocol that can be used in other mouse models to confirm a dependency on basophils, or basophil-derived mediators, for the development of a biological response.