Comparative Investigation of Enzymatic Digestion Techniques for the Preparation of Single Cell Suspensions from Murine Lung Tissue
DONG Xinyi, XIE Nanxi, XU Yudong, WANG Yu, YANG Yongqing*, CHEN Yanjiao*
The present study compares the impact of two types of collagenase enzymes and the combined use of Percoll cell isolation solution on the proportion, cell activity, and quantity of lymphocytes in single cell suspensions derived from mouse lung tissue. These findings provide valuable technical insights for pretreatment in single cell sequencing. Twelve C57BL/6 mice were randomly assigned to four groups, namely the type I collagenase digestion group (G1), type I collagenase digestion+Percoll cell isolation group (G2), type IV collagenase digestion group (G3), and type IV collagenase digestion+Percoll cell isolation group (G4). The cell suspension of lung tissue obtained using these methods was morphologically observed under a microscope, photographed, and quantified. Subsequently, flow cytometry was employed to determine the percentage and number of single cells, viable cells, lymphocytes, as well as the proportion and number of different immune cell subsets. The results showed that no statistically significant difference was found between the two types of collagenase regarding cell dissociation efficiency in mouse lung tissue. However, the G1 group showed a significantly greater abundance of total lung cells than that observed in the G3 group (P<0.05). Moreover, when adjusting for an equivalent cell count across groups, both the G2 and G4 groups exhibited notably higher yields of single cells, viable cells, CD45+ , CD3+ T, CD8+ T and NK cells compared to those seen in either the G1 or G3 groups (P<0.05). Nonetheless, no substantial advantage was noted concerning counts for CD4+ T, CD11c+ , and NKT cells. A higher total cell count was achieved through type I collagenase digestion, while type IV collagenase yielded a greater proportion of lymphocytes. Treatment with Percoll cell separation solution in G2 and G4 groups enables isolation and acquisition of more living single cells, CD45+ cells, CD3+ T cells, CD8+ T cells, and NK cells. These findings provide valuable technical insights for future pretreatment of lung tissue for single cell sequencing.
CN
EN