Linear DNA elution dose response curves obtained in CHO cells with non-unwinding filter elution after appropriate selection of the lysis conditions.
Linear DNA elution dose response curves obtained in CHO cells with non-unwinding filter elution after appropriate selection of the lysis conditions.
复制标题
在适当选择裂解条件后，使用非解旋过滤器洗脱在 CHO 细胞中获得线性 DNA 洗脱剂量响应曲线。
DOI：
10.1080/09553008914550621
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发表时间：
1989
影响因子：
2.6
通讯作者：
Iliakis,G
中科院分区：
文献类型：
--
作者：
Okayasu,R;Iliakis,G
在指数生长、平台期和同步s期CHO细胞的非缠绕条件下，研究了洗涤剂类型、pH和裂解温度对DNA洗脱剂量的影响。与十二烷基硫酸钠（SDS）相比，n -十二烷基肌氨酸钠（NLS）溶解提高了DNA洗脱率，在相同的吸收辐射剂量下，DNA洗脱率更高。洗脱量的增加导致DNA洗脱剂量-反应曲线的肩宽减小，但对最终斜率没有显著影响。在NLS或SDS裂解期间，在高温（60°C）下孵育1小时，进一步增加DNA洗脱。在这些条件下，指数生长、平台期或同步s期细胞获得了小肩或零肩的DNA洗脱剂量-反应曲线。当SDS裂解液的pH值从9·6调整到7·6时，DNA洗脱量减少到对照组的50%左右。在室温裂解的细胞和在60°C裂解的细胞中观察到这种效应。当NLS用于裂解时，类似的pH降低不会改变任一裂解温度下的DNA洗脱剂量-响应曲线。基于这些结果，我们认为在DNA洗脱剂量-反应曲线中观察到的肩部反映了DNA与相关蛋白的部分分离。因此，在非解绕条件下观察到的DNA过滤器洗脱行为与DNA dsb诱导的直接和无条件的相关性可能并不总是合理的。当洗脱结果用于建立DNA dsb诱导水平与细胞杀伤之间的相关性时，需要谨慎。
The effect of detergent type, pH and temperature during lysis on the DNA elution dose response was studied under non-winding conditions in exponentially growing, plateau-phase and synchronous S-phase CHO cells. Lysis with sodium-N-laurylsarcosine (NLS) increased the DNA elution rate and resulted in higher DNA elution for the same absorbed radiation dose than lysis with sodium dodecyl sulphate (SDS). This increase in elution caused a reduction in the shoulder width of the DNA elution dose-response curve, but did not significantly affect the final slope. One hour incubation at elevated temperatures (60°C) during lysis either with NLS or SDS further increased DNA elution. Under these conditions DNA elution dose-response curves with a small or zero shoulder were obtained with exponentially growing, plateau-phase or synchronized S-phase cells. DNA elution was reduced to about 50 per cent of the controls when the pH of the SDS lysis solution was adjusted from 9·6 to 7·6. This effect was observed in cells that were lysed at room temperature, as well as in cells lysed at 60°C. When NLS was used for lysis, a similar reduction in pH did not alter the DNA elution dose-response curve at either lysis temperature. Based on these results it is suggested that the shoulder observed in the DNA elution dose-response curve reflects partial separation of DNA from associated proteins. A direct and unconditional correlation of the DNA filter elution behaviour, as observed under non-unwinding conditions, with the induction of DNA dsb may thus not always be justified. Caution is required when elution results are used to establish correlations between the level of induction of DNA dsb and cell killing.
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DOI：
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发表时间：
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期刊：
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影响因子：
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