Optimisation of bronchoalveolar lavage technique for isolating alveolar macrophages in mice

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Bronchoalveolar lavage (BAL) is a widely used technique to collect immune cells from the lungs, with alveolar macrophages (AMs) being the most prevalent cells in BAL fluid. AMs are vital for maintaining lung homeostasis and providing immune defence against airborne pathogens. However, in murine models, BAL procedures usually yield low numbers of AMs, thus limiting experimental design, especially when high cell counts are needed. Objective — to optimise the BAL technique in mice to maximise the collection of AMs. Materials and methods. Young and older BALB/c mice were used in the study. Bronchoalveolar lavage was performed following the method of Luckow and Lehmann (2021) with modifications. Statistical analysis was done using the Mann‑Whitney U test, with a significance level set at p<0.05. Results. Female BALB/c mice of different ages were chosen due to the frequency of their use as models of pulmonary diseases. A simplified method described by Luckow and Lehmann (2021), which avoids tracheotomy by using peroral cannula insertion, was employed. The protocol was modified by securing the cannula with a ligature to prevent BAL fluid leakage in older mice. To reduce mechanical stress on alveoli, a buffer volume of 0.6 mL was used, and the study compared two buffer variants: one at room temperature without EDTA, and another heated to 37°C with EDTA. The pre‑heated buffer with EDTA significantly increased BAL cell yields in all mice groups, confirming the importance of these optimisations for higher cell recovery. Conclusions. Our modified bronchoalveolar lavage protocol includes securing the trachea with a ligature to prevent BAL fluid leakage, reducing lavage volume to 0.6 mL to minimise lung damage, and using a 37°C solution with EDTA for improved AM recovery rates. Further studies are needed to explore the significance of other buffer components for BAL protocol optimisation, the possible age‑related differences in AM isolation in male BALB/c mice, and the strain‑specific features of the BAL technique.