Adipose tissue-derived stromal cells are promising candidates investigating the stem cell-related treatment. However, their proportion and utility in the human body decline with time, rendering stem cells incompetent to complete repair processes in vivo. The involvement of circRNAs in the aging process is poorly understood. Rat subcutaneous adipose tissue from 10-week-old and 27-month-old rats were used for hematoxylin and eosin (H and E) staining, TUNEL staining, and circRNA sequencing. Rat adipose tissue-derived stromal cells were cultured and overexpressed with circ-ATXN2. Proliferation was examined using xCELLigence real-time cell analysis, EdU staining, and cell cycle assay. Apoptosis was induced by CoCl2 and examined using flow cytometry. RT-PCR assay and Oil Red O staining were used to measure adipogenesis at 48 h and 14 days, respectively. H and E staining showed that the diameter of adipocytes increased; however, the number of cells decreased in old rats. TUNEL staining showed that the proportion of apoptotic cells was increased in old rats. A total of 4,860 and 4,952 circRNAs was detected in young and old rats, respectively. Among them, 67 circRNAs exhibited divergent expression between the two groups (fold change ≥2, p ≤ 0.05), of which 33 were upregulated (49.3%) and 34 were downregulated (50.7%). The proliferation of circ-ATXN2-overexpressing cells decreased significantly in vitro, which was further validated by xCELLigence real-time cell analysis, EdU staining, and cell cycle assay. Overexpression of circ-ATXN2 significantly increased the total apoptotic rate from 5.78 ± 0.46% to 11.97 ± 1.61%, early apoptotic rate from 1.76 ± 0.22% to 5.50 ± 0.66%, and late apoptosis rate from 4.02 ± 0.25% to 6.47 ± 1.06% in adipose tissue-derived stromal cells. Furthermore, in circ-ATXN2-overexpressing cells, RT-PCR assay revealed that the expression levels of adipose differentiation-related genes PPARγ and CEBP/α were increased and the Oil Red O staining assay showed more lipid droplets. Our study revealed the expression profile of circRNAs in the adipose tissue of old rats. We found a novel age-related circular RNA—circ-ATXN2—that inhibits proliferation and promotes cell death and adipogenesis in rat adipose tissue-derived stromal cells.

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.

Flow cytometric sorting is a vital tool in biological research and clinical diagnostics.Theoretically, a high-speed jet-in-air sorter is a fluorescent-activated cell sorting (FACS) sorter that ideally processes cells with high purity, yield and viability. However, high-speed jet-in-air sorting is a complex process due to its inherent requirements for high fluidic stability and electronic and timing precision. Here, we report that an additional manual correction of Drop Delay leads to improved cell yield. Adding 2% FBS to the loading buffer had no significant effect on the fate of sorted cells in 4 hours. However, the addition of a suitable concentration of FBS/BSA in the collecting buffer resulted in a notable increase in cell count and proliferation and a significant decrease in cell apoptosis for cell lines and primary cells. Moreover, the level of gene expression remained steady in the 5% FBS collecting buffer. In summary, here we demonstrate techniques that can be easily followed to refine sorted yields of healthy cells.

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