Evaluation of alternative methods to prepare porcine adipocytes for measurement with an electronic particle number and size determination apparatus.

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.