EVALUATION OF ALTERNATIVE METHODS TO PREPARE ADIPOCYTES FOR MEASUREMENT WITH AN ELECTRONIC PARTICLE NUMBER AND SIZE DETERMINATION APPARATUS

Authors: FAKLER, TIMOTHY - BAYLOR COLL OF MED; MERSMANN, HARRY; SMITH, OBRIAN - BAYLOR COLL OF MEDICINE; MCNEEL, RONALD - BAYLOR COLL OF MEDICINE

Submitted to: Journal of Animal Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/29/1996
Publication Date: N/A
Citation: N/A

Interpretive Summary: Scientific experiments with fat tissue may involve figuring out the number of fat cells and cell size. There are two ways to do this: the microscopic approach and the size instrumental approach. The latter is often preferred because it can evaluate thousands of particles (vs. 50-300), but the standard fixative (used to chemically treat and preserve cells' integrity during handling) is toxic and hazardous to use. We evaluated a number of alternative, less toxic methods to prepare pig fat cells, including glutaraldehyde and formaldehyde, but none of them worked. We then looked at storage methods to keep fixed cells, and again the standard is osmium. Osmium was still the best way to do it, but storage in saline was marginally acceptable.

Technical Abstract: 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 approache to determine adipocyte size and number; microscopic methods use embedded sections, frozen sections, or isolated cells and electronic particle number, and size instrumental methods use adipocytes released from fixed tissue fragments or adipocytes fixed after isolation. The advantage of the instrumental 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 porcine adipocytes appear to be much more susceptible to breakage using these procedures than rat adipocytes. We also added urea or or Triton X-100 to glutaraldehyde - and osmium-fixed cells to decrease clumping and adhesion of individual cells; none of these additions were beneficial. Ability to store samples would improve the logistics for these time-consuming analyses.