Protocols for circulating neutrophil depletion in neonatal C57Bl/6 mice

Background Murine neonatal neutrophil depletion strategies have problems with achieving deep circulating neutrophil clearance and accurate residual neutrophil fraction detection. Boivin et al. demonstrated an isotype switch method of achieving profound neutrophil clearance using a combination of anti-Ly6G and anti-rat κ Ig light chain antibodies in adult C57Bl/6 mice, proven by extra- and intracellular Ly6G detection by flow cytometry. We adapted this technique to neonatal mice testing four neutrophil depletion strategies.

Methods Four protocols were tested: P3 Ly6G and P1-3 Ly6G (anti-Ly6G on postnatal days (P) 3 and 1-3 respectively), and P3 Dual and P1-3 Dual (anti-Ly6G and anti-rat κ Ig light chain on P3 and P1-3 respectively). Intracellular and extracellular Ly6G presence was detected using flow cytometry. Isotype control antibodies were used as controls.

Results P1-3 Dual achieved significantly better neutrophil depletion than the P1-3 Ly6G or P3 Ly6G protocols (97% vs 74% and 97% vs 50%, respectively). The P3 Dual protocol alone was enough to achieve significantly better neutrophil clearance (93%) than any of the Ly6G alone protocols. The Ly6G alone protocols led to near-total elimination of extracellular Ly6G, but there was a significant presence of intracellular Ly6G in the CD45+ cell population, which evaded detection by extracellular Ly6G antibody-based detection methods.

Discussion Anti-Ly6G antibody alone is ineffective in producing effective circulating neutrophil clearance in neonatal mice. Dual antibody-based neutrophil depletion strategies achieve>90% clearance in P4 mice. A single day of dual antibody treatment achieves 93% neutrophil depletion in neonatal mice and is an alternative to daily injections.

Summary Sentence Dual antibody-based neutrophil depletion effectively induces circulating neutrophil clearance in neonatal mice.