在 CYRIS ®分析平台上使用固定化悬浮细胞进行体外线粒体解偶联剂分析和洗脱

IN-VITRO MITOCHONDRIAL UNCOUPLER ASSAY PLUS WASHOUT WITH

IMMOBILIZED SUSPENSION CELLS ON THE CYRIS®ANALYSIS PLATFORM

作者：Christina Heichler & Dr. Peter Wolf（2022）

Abstract：

The following protocol describes the preparation, performance, and results of an in-vitro assay to investigate the response of the suspension cell line YAC-1 to treatment and washout of the mitochondrial chain uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) using our automated cell analysis platform CYRIS®. In an effort to perform high quality measurements on suspension cells in our platform with automated fluidics and imaging, which could be a major challenge with suspension cells, we developed a protocol using Cell-Tak Cell and Tissue Adhesive from Corning® to immobilize the suspension cells to the surface of our sensor plates. For optimal immobilization of suspension cells, it is recommended to determine the minimum effective density of Cell-Tak by a preliminary dose-response experiment as previously described in the application note “Performing an experiment to determine the optimal amount of Cell-Tak to immobilize suspension cells for further analysis on the CYRIS platform”. The CYRIS® platform enables the automated examination of the effects of FCCP on suspension cells due to its ability to monitor oxygen consumption rate (OCR), extracellular acidification rate (ECAR) and microscopic images of the cells, label-free, in parallel and real-time. The cells are measured for 2 hours without treatment for a baseline, 6 hours under treatment with different FCCP concentrations (0 nM, 125 nM or 250 nM) and 2 hours after treatment for washout. The suspension cells were effectively immobilized to the surface of our sensor plate throughout the entire experiment. The uncoupling of the respiratory proton gradient by FCCP treatment of suspension cells was reflected by increased OCR and ECAR due to the associated additional effort of the cell to stabilize this gradient. The effect of FCCP was not permanent and could be washed

文章摘要：

以下案例讲述了体外测定的制备、性能和结果。使用CYRIS®自动化细胞分析平台研究了悬浮细胞系 YAC-1 对线粒体链解偶联剂羰基氰化物-对三氟甲氧基苯腙 (FCCP) 的处理和冲洗的反应 。为了通过自动化流体和成像对仪器中的悬浮细胞进行高质量测量，这可能是悬浮细胞的主要挑战。我们开发了一种使用 Corning® 的 Cell-Tak Cell 和 Tissue Adhesive 来固定悬浮细胞的方案到传感器板的表面。为了悬浮细胞的最佳固定，建议通过初步剂量反应实验来确定 Cell-Tak 的最小有效密度，如先前在应用说明“执行实验以确定 Cell-Tak 的最佳量以固定悬浮液”中所述用于在 CYRIS 平台上进一步分析的细胞”。

CYRIS® 平台能够自动检测 FCCP 对悬浮细胞的影响，因为它能够监测耗氧率 (OCR)、细胞外酸化率 (ECAR) 和细胞显微图像，无需标记，实时监测。细胞在未处理的情况下测量 2 小时，然后在不同 FCCP 浓度（0 nM、125 nM 或 250 nM）下测量 6 小时，并在处理后 2 小时测量清除。在整个实验过程中，悬浮细胞被有效地固定在传感器板表面。由于细胞为稳定该梯度而付出的额外努力，因此 OCR 和 ECAR 的增加反映了悬浮细胞的 FCCP 处理对呼吸质子梯度的解偶联。 FCCP的效果不是永久的，可以洗掉。

点击链接即可查看和下载文章：https://www.cyris.bio/wp-content/uploads/2022/12/20220718-Suspension-cells_FCCP-treatment-and-washout_Application-Note_my.pdf

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