Improved Strategy for Jet-in-air Cell Sorting With High Purity, Yield, Viability and Genome Stability

Abstract

Background: The enrichment of viable cells is an essential step in single-cell sequencing for clinical and biotechnology research applications. A high-end jet-in-air sorter is a fluorescent-activated cell sorting sorter that ideally processes cells with high purity, yield and viability. We want to improve the strategy for jet-in-air cell sorting to get more effective cells, and ensure the downstream experiments. Method: In this study, to achieve high-purity, high-yield, high-viability cell sorting, we explored three aspects. First, we manually adjusted the drop delay through the beads experiment to make it more accurate and to ensure the highest yield in theory, thereby avoiding the influence of the instrument and operator. Second, the effects on cell apoptosis caused by adding 2% fetal bovine serum to the loading buffer were examined. Third, the effects on cell count, apoptosis, proliferation and gene expression caused by employing FBS and bovine serum albumin in the collecting buffer were analyzed. Statistical analysis was performed with GraphPad Prism 6 software. Significance was set at p < 0.05.Result: Our data showed that an additional manual correction of drop delay could lead to an improved cell yield of 98.28%±3.79%. Adding FBS (2%) to the loading buffer had no significant effect on the fate of sorted cells in 4 hours. However, employing a suitable concentration of FBS/BSA in the collecting buffer mediated a notable increase in cell count from 50% to ~100%, a significant decrease in cell apoptosis from ~40% to less than 10% for cell lines and ~70% to less than 40% for CD45+ primary cells from mouse spleen. And a clear increase in cell proliferation at 48 h after sorting for jurkat cells. Moreover, the level of gene expression remained steady in the 5% FBS collecting buffer. Conclusions: Our manuscript demonstrates techniques that can be easily followed to refine sort yields of healthy cells. Fluorescent-activated cell sorting users could easily obtain enough target cells with great viability and need not to worry about any problems related to collecting or loading buffer. Furthermore, adding extra expensive FBS in collecting or loading buffer to boost yield is no longer necessary. In all, this will make the downstream experiments smoother.

Rapid Isolation of Single Cells from Mouse and Human Teeth

“…Stopping the dissociation process is ensured by diluting the Collagenase P in Wash solution (2% FBS in HBSS). It has been shown that adding FBS increases cell viability and the final number of cells after sorting18 .2. Using a round-shaped scalpel blade no.…”