optimization-flow-cytometric-sorting-parameters-for-high-throughput

Optimization of Flow Cytometric Sorting Parameters for High-Throughput Isolation and Purification of Small Extracellular Vesicles

Small extracellular vesicles (sEV) can be released from all cell types and carry protein, DNA, and RNA. Signaling molecules serve as indicators of the ...

Zhejiang University School of Medicine

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for  autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition).

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 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 h. 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.

Improved strategy for jet-in-air cell sorting with high purity, yield, viability, and genome stability.

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 . 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,  ≤ 0.05), of which 33 were upregulated (49.3%) and 34 were downregulated (50.7%). The proliferation of circ-ATXN2-overexpressing cells decreased significantly , 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  and  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.

Chun Guo

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Zhejiang University School of Medicine

Optimization of Flow Cytometric Sorting Parameters for High-Throughput Isolation and Purification of Small Extracellular Vesicles

Chun Guo

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Zhejiang University School of Medicine

Optimization of Flow Cytometric Sorting Parameters for High-Throughput Isolation and Purification of Small Extracellular Vesicles

Core Facilities