Laboratory of Natural Products, Institute of Bioscience, Universiti Putra Malaysia, 43400, Serdang, Malaysia.
Laboratory of Natural Products, Institute of Bioscience, Universiti Putra Malaysia, 43400, Serdang, Malaysia; Department of Food Science, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400, Serdang, Malaysia.
Anal Biochem. 2019 Jul 1;576:20-32. doi: 10.1016/j.ab.2019.04.001. Epub 2019 Apr 7.
The variation in the extracellular metabolites of RAW 264.7 cells obtained from different passage numbers (passage 9, 12 and 14) was examined. The impact of different harvesting protocols (trypsinization and scraping) on recovery of intracellular metabolites was then assessed. The similarity and variation in the cell metabolome was investigated using H NMR metabolic profiling modeled using multivariate data analysis. The characterization and quantification of metabolites was performed to determine the passage-related and harvesting-dependent effects on impacted metabolic networks. The trypsinized RAW cells from lower passages gave higher intensities of most identified metabolites, including asparagine, serine and tryptophan. Principal component analysis revealed variation between cells from different passages and harvesting methods, as indicated by the formation of clusters in score plot. Analysis of S-plots revealed metabolites that acted as biomarkers in discriminating cells from different passages including acetate, serine, lactate and choline. Meanwhile lactate, glutamine and pyruvate served as biomarkers for differentiating trypsinized and scraped cells. In passage-dependent effects, glycolysis and TCA cycle were influential, whereas glycerophospholipid metabolism was affected by the harvesting method. Overall, it is proposed that typsinized RAW cells from lower passage numbers are more appropriate when conducting experiments related to NMR metabolomics.
考察了不同传代数（第 9、12 和 14 代）RAW 264.7 细胞的细胞外代谢物的变化。然后评估了不同收获方案（胰蛋白酶消化和刮取）对内源代谢物回收的影响。使用基于多变量数据分析的 H NMR 代谢组学模型研究了细胞代谢组的相似性和变异性。对代谢物进行了特征描述和定量，以确定对受影响代谢网络有影响的传代和收获依赖性效应。较低传代数的胰蛋白酶化 RAW 细胞给出了大多数鉴定出的代谢物（包括天冬酰胺、丝氨酸和色氨酸）的更高强度。主成分分析显示了不同传代和收获方法的细胞之间的差异，这表现为得分图中簇的形成。S 图分析显示了作为区分不同传代细胞的生物标志物的代谢物，包括乙酸盐、丝氨酸、乳酸盐和胆碱。同时，乳酸盐、谷氨酰胺和丙酮酸可作为区分胰蛋白酶化和刮取细胞的生物标志物。在传代依赖性效应中，糖酵解和 TCA 循环是有影响的，而甘油磷脂代谢受收获方法的影响。总体而言，建议在进行与 NMR 代谢组学相关的实验时，使用来自较低传代数的胰蛋白酶化 RAW 细胞。
