Parabolic growth patterns in 96-well plate cell growth experiments.
Parabolic growth patterns in 96-well plate cell growth experiments.
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96 孔板细胞生长实验中的抛物线生长模式。
DOI：
10.1007/s11626-999-0071-z
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Greco,WR
中科院分区：
文献类型：
--
作者：
Faessel,HM;Levasseur,LM;Slocum,HK;Greco,WR
在为普遍使用的体外细胞生长抑制试验研究抗癌药物的常规使用做准备的过程中，我们对该试验的统计特性进行了表征，并发现了一些令人惊讶的结果。发现了抛物线型井到井细胞生长模式，这可能深刻影响抗癌和其他药物的常规生长抑制研究的结果。4株人卵巢癌细胞株A2780/WT、A2780/DX5、A2780/DX5B和A121和1株人回盲腺癌细胞株HCT-8接种于塑料96孔板中，用12通道移液管，不加药物，培养1~5d，用洗板机洗涤细胞，用硫代罗丹明B(SRB)染色的细胞用平板阅读器测量染料吸光度。对重复数据进行方差模型拟合，以确定异方差误差结构的性质。指数增长模型适用于数据，以估计每个细胞系的加倍时间。用多项式模型对96孔板的10孔板的数据进行了拟合，以探索不同区域孔板中细胞生长的不均匀性。分析中的每一个步骤都被检查了精密度、模式和潜在的变异原因。井中水分的差异蒸发可能是系统的井间细胞生长模式的主要因素，但不是唯一的因素。由于抛物线生长模式的根本原因没有得到最终的发现，因此制定了生长分析的随机化步骤。
In preparing for the routine use of the ubiquitous in vitro cell growth inhibition assay for the study of anticancer agents, we characterized the statistical properties of the assay and found some surprising results. Parabolic well-to-well cell growth patterns were discovered, which could profoundly affect the results of routine growth inhibition studies of anticancer and other agents. Four human ovarian cell lines, A2780/WT, A2780/DX5, A2780/DX5B, and A121, and one human ileocecal adenocarcinoma cell line, HCT-8, were seeded into plastic 96-well plates with a 12-channel pipette, without drugs, and grown from 1–5 d. The wells were washed with a plate washer, cells stained with sulforhodamine B (SRB), and dye absorbance measured with a plate reader. Variance models were fit to the data from replicates to determine the nature of the heteroscedastic error structure. Exponential growth models were fit to data to estimate doubling times for each cell line. Polynomial models were fit to data from 10-plate stacks of 96-well plates to explore nonuniformity of cell growth in wells in different regions of the stacks. Each separate step in the assay was examined for precision, patterns, and underlying causes of variation. Differential evaporation of water from wells is likely a major, but not exclusive, contributor to the systematic well-to-well cell growth patterns. Because the fundamental underlying causes of the parabolic growth patterns were not conclusively found, a randomization step for the growth assay was developed.