Seidl J, Knuechel R, Kunz-Schughart L A
Institute of Pathology, University of Regensburg, Germany.
Cytometry. 1999 Jun 1;36(2):102-11. doi: 10.1002/(sici)1097-0320(19990601)36:2<102::aid-cyto3>3.3.co;2-4.
Over the past decade, cell separation technology has become an important tool in various fields of cell biology allowing for the analysis or subsequent cultivation of specific cell subsets. The objective of the present study was to evaluate if the established sorting techniques fluorescence-activated (FACS) and magnetic cell separation (MACS) affect cell membrane physiology in order to define the most non-perturbing application for the separation of tumor and stromal cells.
Membrane physiology was monitored in single cell suspensions of adherently grown BT474 breast tumor cells and N1 normal skin fibroblasts using flow cytometry. Cell membrane integrity was evaluated by propidium iodide (PI) staining. Microviscosity within the lipophilic membrane layer was determined by a monomer/excimer method utilizing pyrene decanoic acid, membrane potential measurements were carried out using the fluorescence indicator DiBAC4(3), and Annexin-V-staining reflected transversal membrane asymmetry, and an altered phospholipid distribution.
Not only the number of preparative cycles prior to cell separation but also the sort conditions during FACS resulted in loss of membrane integrity of a certain cell fraction. If these PI-positive cells were excluded from further analysis, neither MACS nor FACS affected membrane microviscosity while a clear hyperpolarization in both cell types after MACS resulting from exposure to the ferromagnetic matrix of the depletion column and the inhomogeneous magnetic field was shown. In addition, cell sorting of BT474 tumor cells by MACS and FACS was accompanied by the generation of an Annexin-V-positive/PI-negative cell fraction with altered phospholipid distribution. Data were discussed with regard to the sort-induced "stress" conditions such as exposure to hydrodynamic forces or magnetic fields.
Both separation procedures modify cell membrane with neither technique being physiologically preferable for subsequent analysis or recultivation of the sorted cells.
在过去十年中，细胞分离技术已成为细胞生物学各个领域的重要工具，可用于特定细胞亚群的分析或后续培养。本研究的目的是评估既定的分选技术——荧光激活细胞分选（FACS）和磁性细胞分离（MACS）是否会影响细胞膜生理学，以便确定用于分离肿瘤细胞和基质细胞的最微创应用。
使用流式细胞术监测贴壁生长的BT474乳腺肿瘤细胞和N1正常皮肤成纤维细胞的单细胞悬液中的膜生理学。通过碘化丙啶（PI）染色评估细胞膜完整性。利用癸酸通过单体/激基缔合物法测定亲脂性膜层内的微粘度，使用荧光指示剂DiBAC4(3)进行膜电位测量，膜联蛋白V染色反映横向膜不对称性以及磷脂分布的改变。
不仅细胞分离前的制备循环次数，而且FACS期间的分选条件都会导致一定比例的细胞失去膜完整性。如果将这些PI阳性细胞排除在进一步分析之外，MACS和FACS均不影响膜微粘度，而MACS后两种细胞类型均出现明显的超极化，这是由于暴露于耗尽柱的铁磁基质和不均匀磁场所致。此外，通过MACS和FACS对BT474肿瘤细胞进行细胞分选时，会产生磷脂分布改变的膜联蛋白V阳性/PI阴性细胞部分。针对分选诱导的“应激”条件，如暴露于流体动力或磁场，对数据进行了讨论。
两种分离方法都会改变细胞膜，对于分选后细胞的后续分析或再培养，两种技术在生理学上都不是更可取的。
