Toxic effects of trace phenol/guanidine isothiocyanate (P/GI) on cells cultured nearby in covered 96-well plates.
Toxic effects of trace phenol/guanidine isothiocyanate (P/GI) on cells cultured nearby in covered 96-well plates.
复制标题
DOI：
10.1186/s12896-022-00766-2
复制
发表时间：
2022-11-25
影响因子：
3.5
通讯作者：
Hirsch, Emmet
中科院分区：
文献类型：
--
作者：
Snedden, Madeline;Singh, Lavisha;Kyathanahalli, Chandrashekara;Hirsch, Emmet
苯酚和异硫氰酸胍的混合物（“P/GI”，TRIzol™和类似产品的主要成分）通常用于从单个样本中分离RNA、DNA和蛋白质。在组织培养中生长的细胞的时程实验中，通常顺序收获重复的威尔斯孔并进行比较，假设孔内裂解和P/GI的完全抽吸对附近威尔斯孔中的继续培养没有影响。为了验证这一假设，我们研究了RAW 264.7细胞（一种永生化小鼠巨噬细胞系）的形态和功能，该细胞在覆盖的96孔板中培养4、8或24 h，与单个对照孔或P/GI沉积并立即完全吸出的孔相距不同距离。在细胞形态（起泡、细胞质破坏和细胞内囊泡积聚）、细胞功能（培养基pH值）以及炎症相关基因（TNFα）和自噬相关基因（Lc 3b）表达中观察到因接近含有微量残留P/GI的单个孔而导致的时间和距离依赖性破坏。抗凋亡基因Mcl 1、促凋亡基因Hrk和P/GI未暴露对照培养物中均无转录变化。在P/GI附近孵育的LPS刺激的细胞具有较低的细胞因子IL 6表达。这些影响早在4小时的暴露和距离P/GI暴露井多达3个井单位。在覆盖的组织培养板中暴露于痕量残留量的P/GI导致细胞形态和功能在短至4小时内发生实质性破坏，可能是通过诱导自噬而不是凋亡。当计划在多孔覆盖的组织培养板中进行时程实验时，应考虑这种现象。在线版本包含补充材料，可通过10.1186/s12896-022-00766-2获得。
A mixture of phenol and guanidine isothiocyanate (“P/GI”, the principal components of TRIzol™ and similar products) is routinely used to isolate RNA, DNA, and proteins from a single specimen. In time-course experiments of cells grown in tissue culture, replicate wells are often harvested sequentially and compared, with the assumption that in-well lysis and complete aspiration of P/GI has no effect on continuing cultures in nearby wells. To test this assumption, we investigated morphology and function of RAW 264.7 cells (an immortalized mouse macrophage cell line) cultured in covered 96-well plates for 4, 8, or 24 h at varying distances from a single control well or a well into which P/GI had been deposited and immediately aspirated completely. Time- and distance-dependent disruptions resulting from proximity to a single well containing trace residual P/GI were seen in cell morphology (blebbing, cytoplasmic disruption, and accumulation of intracellular vesicles), cell function (pH of culture medium), and expression of genes related to inflammation (Tnfα) and autophagy (Lc3b). There was no transcriptional change in the anti-apoptotic gene Mcl1, nor the pro-apoptotic gene Hrk, nor in P/GI-unexposed control cultures. LPS-stimulated cells incubated near P/GI had lower expression of the cytokine Il6. These effects were seen as early as 4 h of exposure and at a distance of up to 3 well units from the P/GI-exposed well. Exposure to trace residual quantities of P/GI in covered tissue culture plates leads to substantial disruption of cell morphology and function in as little as 4 h, possibly through induction of autophagy but not apoptosis. This phenomenon should be considered when planning time-course experiments in multi-well covered tissue culture plates. The online version contains supplementary material available at 10.1186/s12896-022-00766-2.
登录
查看更多内容
影响因子：
3.2
作者：
Jung, Ha-Na;Zerin, Tamanna;Kim, Yong-Sik
通讯作者：
Kim, Yong-Sik
影响因子：
6.1
作者：
Tolosa L;Martínez-Sena T;Schimming JP;Moro E;Escher SE;Ter Braak B;van der Water B;Miranda MA;van Vugt-Lussenburg BMA;Castell JV
通讯作者：
Castell JV
影响因子：
32.4
作者：
Pearce EL;Pearce EJ
通讯作者：
Pearce EJ
影响因子：
8.8
作者：
Girnius N;Davis RJ
通讯作者：
Davis RJ
DOI：
10.1615/critreveukargeneexpr.v20.i2.10
发表时间：
2010
影响因子：
1.6
作者：
Parameswaran N;Patial S
通讯作者：
Patial S