Fakler T, O'Brian Smith E, McNeel R L, Mersmann H J
USDA/ARS Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030, USA.
J Anim Sci. 1996 Oct;74(10):2385-93. doi: 10.2527/1996.74102385x.
Experimental investigations with mammalian adipose tissue require a determination of adipocyte number as a basis for expression of metabolic and growth data. Determination of cell size is also important in adipose tissue because the fivefold or greater variation in adipocyte diameter in most growing and adult mammals precludes simple determination of cell number to interpret the biological observations. There are two approaches to determine adipocyte size and number: microscopic methods and electronic particle counter methods. Microscopic methods use embedded sections, frozen sections, or isolated cells, whereas electronic particle number and size instrumental methods use adipocytes released from fixed tissue fragments or adipocytes fixed after isolation. The advantage of the electronic approach is that it evaluates thousands of particles, although the standard fixative, osmium, is quite toxic. Consequently, we evaluated a number of alternative fixation methods to prepare isolated porcine adipocytes for number and size determination by electronic instrumentation. Fixation in 3, 4, or 5% glutaraldehyde or in 4% formaldehyde were not acceptable procedures for porcine adipocytes. The 4% glutaraldehyde fixation procedure was acceptable for isolated rat adipocytes (Stewart and Kaplan, 1993); porcine adipocytes seem to be much more susceptible to breakage using these procedures than rat adipocytes. We also added urea or Triton X-100 to glutaraldehyde- and osmium-fixed cells to decrease clumping and adhesion of individual cells; none of these additions was beneficial. Ability to store samples would improve the logistics for these time-consuming analyses. Samples of osmium-fixed adipocytes were stored in osmium, in .9% NaCl (saline) after removal of osmium, in 8 M urea after osmium removal with saline, or in .01% Triton X-100 after osmium removal with saline. Storage in urea or Triton was inappropriate because of irreversible clumping of individual cells. Storage in osmium was acceptable for at least 30 d. and storage in saline was marginally acceptable. The variability of the size determination process for osmium-fixed adipocytes was evaluated.
对哺乳动物脂肪组织进行实验研究需要确定脂肪细胞数量，以此作为代谢和生长数据表达的基础。在脂肪组织中，细胞大小的测定也很重要，因为在大多数生长中的和成年哺乳动物中，脂肪细胞直径有五倍或更大的差异，这使得简单地通过测定细胞数量来解释生物学观察结果变得不可能。有两种方法来确定脂肪细胞的大小和数量：显微镜方法和电子粒子计数器方法。显微镜方法使用包埋切片、冷冻切片或分离的细胞，而电子粒子数量和大小测量方法则使用从固定组织碎片中释放的脂肪细胞或分离后固定的脂肪细胞。电子方法的优点是它可以评估数千个粒子，尽管标准固定剂锇毒性很大。因此，我们评估了许多替代固定方法，以制备分离的猪脂肪细胞，用于通过电子仪器测定数量和大小。在3%、4%或5%的戊二醛或4%的甲醛中固定，对于猪脂肪细胞来说不是可接受的方法。4%戊二醛固定程序对于分离的大鼠脂肪细胞是可接受的（Stewart和Kaplan，1993）；使用这些程序时，猪脂肪细胞似乎比大鼠脂肪细胞更容易破裂。我们还向戊二醛和锇固定的细胞中添加尿素或曲拉通X-100，以减少单个细胞的聚集和粘附；这些添加物都没有效果。储存样品的能力将改善这些耗时分析的后勤工作。锇固定的脂肪细胞样品储存在锇中、去除锇后储存在0.9%氯化钠（生理盐水）中、用生理盐水去除锇后储存在8M尿素中或用生理盐水去除锇后储存在0.01%曲拉通X-100中。储存在尿素或曲拉通中不合适，因为单个细胞会发生不可逆的聚集。储存在锇中至少30天是可接受的，储存在生理盐水中勉强可接受。我们评估了锇固定的脂肪细胞大小测定过程的可变性。
