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Flow cytometric analysis from fish samples stored at low, ultra-low and cryogenic temperatures
Cryobiology ( IF 2.7 ) Pub Date : 2020-08-01 , DOI: 10.1016/j.cryobiol.2020.06.004
George Shigueki Yasui 1 , Rafaela Manchin Bertolini 2 , Lucia Suárez-López 2 , Pedro Porfírio Xavier 3 , Paulo Sérgio Monzani 4 , Nivaldo Ferreira do Nascimento 1 , Antonio Leão Castilho 5 , Laura Satiko Okada Nakaghi 6 , Silvio Carlos Alves Dos Santos 7 , José Augusto Senhorini 1
Affiliation  

Flow cytometry is a valuable tool in biomedical and animal sciences. However, equipment used for such analysis presents limitations at field conditions, suggesting then preservation procedures for future analysis at laboratory conditions. In this study, freezing at low (-20 °C), ultra-low (-80 °C) and cryogenic temperatures (-196 °C, i.e. liquid nitrogen) were used as preservation procedures of fish tissue. Samples were maintained in 0.9% NaCl or lysing solution, and stored at the temperatures above for 0 (fresh control), 60, 120 and 180 days of storage. After storage, the samples were thawed and proceeded to flow cytometric analysis. Storage at low temperatures (-20 °C), both in lysing and 0.9% NaCl, exhibited poor results when analyzed after 60, 120 and 180 days, showing noisy peaks, deviation in the DNA content and absence of peaks. Ultralow (-80 °C) and cryogenic (-196 °C) temperatures, both in lysing solution and 0.9% NaCl, showed good results and high quality of histograms. Both storage procedures gave similar histograms and DNA content in comparison with control group (fresh) even after 60, 120 and 180 days of storage, exhibiting the main peak at 2C content from diploid cells and a secondary peak at 4C derived from dividing cells. In conclusion, samples may be stored for 180 days at -80 °C and -196 °C in both, 0.9% NaCl or lysing solution. As cryogenic temperatures in liquid nitrogen permits indefinite storage, this procedure should be used for long-term preservation.

中文翻译:

对储存在低温、超低温和低温下的鱼样品进行流式细胞术分析

流式细胞术是生物医学和动物科学中的重要工具。然而,用于此类分析的设备在现场条件下存在局限性,这表明了在实验室条件下进行未来分析的保存程序。在这项研究中,低温(-20°C)、超低温(-80°C)和低温(-196°C,即液氮)冷冻被用作鱼类组织的保存程序。将样品保存在 0.9% NaCl 或裂解溶液中,并在上述温度下储存 0(新鲜对照)、60、120 和 180 天。储存后,将样品解冻并进行流式细胞术分析。在低温 (-20 °C) 下储存,无论是溶解性还是 0.9% NaCl,在 60、120 和 180 天后分析时都表现出较差的结果,显示出嘈杂的峰、DNA 含量的偏差和峰的缺失。裂解液和 0.9% NaCl 中的超低 (-80 °C) 和低温 (-196 °C) 温度均显示出良好的结果和高质量的直方图。与对照组(新鲜)相比,即使在储存 60、120 和 180 天后,两种储存程序都给出了相似的直方图和 DNA 含量,显示出来自二倍体细胞的 2C 含量的主峰和来自分裂细胞的 4C 的次要峰。总之,样品可以在 -80 °C 和 -196 °C 下在 0.9% NaCl 或裂解溶液中储存 180 天。由于液氮中的低温允许无限期储存,因此该程序应用于长期保存。与对照组(新鲜)相比,即使在储存 60、120 和 180 天后,两种储存程序都给出了相似的直方图和 DNA 含量,显示出来自二倍体细胞的 2C 含量的主峰和来自分裂细胞的 4C 的次要峰。总之,样品可以在 -80 °C 和 -196 °C 下在 0.9% NaCl 或裂解溶液中储存 180 天。由于液氮中的低温允许无限期储存,因此该程序应用于长期保存。与对照组(新鲜)相比,即使在储存 60、120 和 180 天后,两种储存程序都给出了相似的直方图和 DNA 含量,显示出来自二倍体细胞的 2C 含量的主峰和来自分裂细胞的 4C 的次要峰。总之,样品可以在 -80 °C 和 -196 °C 下在 0.9% NaCl 或裂解溶液中储存 180 天。由于液氮中的低温允许无限期储存,因此该程序应用于长期保存。
更新日期:2020-08-01
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