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 de termination of adipocyte number as a basis for expression of metabolic and growth data. Determination of cell size is also important in adip ose tissue because the fivefold or greater variation in adipocyte diam eter 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 method s and electronic particle counter methods. Microscopic methods use emb edded 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. Co nsequently, we evaluated a number of alternative fixation methods to p repare isolated porcine adipocytes for number and size determination b y electronic instrumentation. Fixation in 3, 4, or 5% glutaraldehyde o r in 4% formaldehyde were not acceptable procedures for porcine adipoc ytes. The 4% glutaraldehyde fixation procedure was acceptable for isol ated rat adipocytes (Stewart and Kaplan, 1993); porcine adipocytes see m 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 sampl es would improve the logistics for these time-consuming analyses. Samp les of osmium-fixed adipocytes were stored in osmium, in .9% NaCl (sal ine) after removal of osmium, in 8 M urea after osmium removal with sa line, or in .01% Triton X-100 after osmium removal with saline. Storag e 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 o f the size determination process for osmium-fixed adipocytes was evalu ated.