Optimization of mass cytometry sample cryopreservation after staining
Optimization of mass cytometry sample cryopreservation after staining
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DOI：
10.1002/cyto.a.23014
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发表时间：
2017-01-01
期刊：
影响因子：
3.7
通讯作者：
Newell, Evan W.
中科院分区：
文献类型：
--
作者：
Sumatoh, Hermi R.;Teng, Karen Wei Weng;Newell, Evan W.
质谱细胞术的出现促进了高度多参数的单细胞分析，从而允许对细胞多样性进行深入评估。虽然这种方法的数据和分析能力得到了很好的描述，但相关的技术和实验障碍仍然存在。设备故障和大规模批次取样等问题可能需要数天的数据采集，这些问题虽小但至关重要，需要采取技术解决方案，特别是在处理珍贵样本时。冷冻保存已经染色的大量细胞计数样品的能力将减轻我们目前使用的样品处理方法所面临的许多技术限制。在这里，我们评估了两个协议的冷冻已经染色和固定的细胞样本，并比较它们与标准样品冷冻染色缓冲液。使用综合的人T细胞染色表型和功能分析面板，并在4周内评估每种标志物的信号强度和可靠性。一般而言，与冷藏相比，冷冻对细胞活力、DNA Ir-嵌入剂和条形码染色的影响最小，并且细胞表面标志物和受体的信号强度不受影响。细胞内细胞因子染色确实显示冷冻后信号强度有所降低，与涉及使用FBS中10%DMSO的方案相比，基于甲醇的方案中的降低更显著。我们的结论是，冷冻已染色的样品悬浮在10%DMSO的FBS是实用和有效的方法来保存已染色的样品时，需要的。(c)2016年国际细胞计数促进学会
The advent of mass cytometry has facilitated highly multi-parametric single-cell analysis allowing for the deep assessment of cellular diversity. While the data and analytical power of this approach are well described, associated technical and experimental hurdles remain. Issues like equipment breakdown and sampling of large-scale batches, which may require multiple days of data acquisition, are minor but critical obstacles that prompt a technical solution, especially when dealing with precious samples. An ability to cryopreserve mass cytometry samples that have already been stained would alleviate numerous technical limitations we face with currently used sample-handling approaches. Here, we evaluated two protocols for freezing of already-stained and fixed cellular samples and compared them with standard sample refrigeration in staining buffer. A comprehensive human T cell staining phenotypic and functional profiling panel was used and the signal intensity and reliability of each marker was assessed over a 4-week period. In general, cellular viability, DNA Ir-Intercalator and barcode staining were minimally affected by freezing compared to refrigeration, and the signal intensities for cell surface markers and receptors were not compromised. Intracellular cytokine staining did show some decreases in signal intensity after freezing, with the decreases more prominent in a methanol-based protocol compared to a protocol involving the use of 10% DMSO in FBS. We conclude that freezing already-stained samples suspended in 10% DMSO in FBS is practical and efficient way to preserve already-stained samples when needed. (c) 2016 International Society for Advancement of Cytometry