Gilbert C S, Parmley R T
Carolinas Medical Center, Department of Pediatric Research, Charlotte, North Carolina 28203, USA.
Anat Rec. 1998 Oct;252(2):254-63. doi: 10.1002/(SICI)1097-0185(199810)252:2<254::AID-AR10>3.0.CO;2-M.
Cell shape and density are critical to the evaluation of neutrophil function and/or activation. Dimethyl sulfoxide-cryofixation-freeze-substitution processing (DCF) instantly preserves cell processes and ultrastructural elements with fewer artifacts than routine chemical fixation with glutaraldehyde and postfixation osmium tetroxide (GO). This study morphometrically examined density-separated neutrophils to assess differences in DCF and GO processing procedures and studied the effect of dimethyl sulfoxide followed by GO fixation (DGO) on morphology. Fifteen consecutive neutrophils were analyzed using computerized planimetry for differences in DCF v. GO treatments (n = 4) and DGO v. GO treatments (n = 4). Cryofixed and DGO-fixed cells were significantly rounder than GO cells which had a more irregular surface with membrane projections. The cell volume of GO cells was 27-30% smaller than in DCF or DGO processing, while the surface area was similar. The increased volume in DCF and DGO cells did not appear to be due to abnormal cell swelling, since membranes, nuclear envelope, and mitochondrial cristae were more intact than in GO cells. Preservation of mitochondria as well as endocytic caveolae with a subplasmalemmal coating was best in DCF samples, moderate in DGO, and poorest in GO. Morphometric data showed that the nuclear compartment was 22% smaller, while the cytoplasm (and its associated compartments) was 29% smaller in GO compared to DCF-processed neutrophils. This was consistent with the more dense cytoplasm in GO cells. Pretreatment of neutrophils with dimethyl sulfoxide (DMSO) resulted in volume preservation and improved the morphology of GO fixation. In summary, DCF appears to be an excellent method for preserving neutrophil membranes and cytoplasmic organelles (particularly mitochondria), and prevents a number of artifacts caused by routine GO fixation. Morphology can also be improved by using DMSO in conjunction with GO.
细胞形状和密度对于评估中性粒细胞功能和/或活化至关重要。二甲基亚砜冷冻固定-冷冻置换处理（DCF）能即时保存细胞突起和超微结构成分，与戊二醛常规化学固定及四氧化锇后固定（GO）相比，产生的伪像更少。本研究通过形态计量学方法检查密度分离的中性粒细胞，以评估DCF和GO处理程序的差异，并研究二甲基亚砜随后进行GO固定（DGO）对形态的影响。使用计算机平面测量法分析了连续的15个中性粒细胞，以比较DCF与GO处理（n = 4）以及DGO与GO处理（n = 4）的差异。冷冻固定和DGO固定的细胞比GO细胞明显更圆，GO细胞表面更不规则且有膜突起。GO细胞的细胞体积比DCF或DGO处理的细胞小27 - 30%，而表面积相似。DCF和DGO细胞中增加的体积似乎并非由于细胞异常肿胀，因为其细胞膜、核膜和线粒体嵴比GO细胞更完整。DCF样本中线粒体以及带有质膜下包被的内吞小窝保存最佳，DGO样本中适中，GO样本中最差。形态计量学数据显示，与DCF处理的中性粒细胞相比，GO处理的中性粒细胞核区小22%，而细胞质（及其相关区室）小29%。这与GO细胞中更致密的细胞质一致。用二甲基亚砜（DMSO）预处理中性粒细胞可保持体积并改善GO固定的形态。总之，DCF似乎是保存中性粒细胞膜和细胞质细胞器（特别是线粒体）的极佳方法，并可防止常规GO固定引起的许多伪像。联合使用DMSO和GO也可改善形态。
