Fig 1: CHIPS binds and inhibits hC5aR1KI murine neutrophils at levels comparable to those with human neutrophils. Quantification of hC5aR1 expression in hC5aR1KI mice showed similar expression levels compared to human leukocytes (Tromp et al., 2018). Furthermore, hC5aR1KI murine neutrophils responded normally to both murine C5a (mC5a) and human C5a as measured by Ca mobilization (Tromp et al., 2018). (A) hC5aR1KI bone marrow neutrophils and human blood neutrophils were isolated and incubated with 3 µg/ml histidine-tagged CHIPS followed by anti-histidine-fluorescein isothiocyanite (FITC) antibodies. Cells were analyzed by flow cytometry and the FITC fluorescent signal was depicted as histograms. (B) As our hC5aR1KI murine model generates mC5a, the assessment of CHIPS inhibition was performed by mC5a stimulation. Bone marrow neutrophils of hC5aR1KI, wild-type (WT) mice and human neutrophils were pre-incubated with CHIPS at the indicated concentration and subsequently stimulated with murine C5a (10-8M). The basal fluorescence level was first measured for each sample before the addition of murine C5a. The C5a-mediated calcium influx was analyzed by flow cytometry using Fluo-4AM. The average Fluo-4AM fluorescent signal was used to calculate CHIPS-mediated inhibition of C5a responses. One experiment representative of two independent experiments is shown.