β-adrenergic receptor binding in crude porcine adipose tissue plasma membranes

Abstract

Methods have been detailed to prepare a crude membrane fraction from isolated porcine adipose tissue cells. Adipocytes were obtained after incubation of 5 g of adipose tissue slices with 4,500 units of a selected lot of collagenase in a total volume of 15 mL at 37 degrees C for 90 min. There was no bovine serum albumin present during cell isolation because albumin did not enhance cell yield or yield of lipolytic activity. Isolated cells were lysed by exposure to hypotonic conditions in the presence of 7.5 mM ethylene glycol tetraacetic acid (EGTA) and .8 mM phenylmethylsulfonyl fluoride (PMSF). A 30,000 x g centrifugal pellet was used as the crude membrane preparation. Binding of tritiated dihydroalprenolol (DHA), a beta-adrenergic antagonist, was measured in the presence of 7.5 mM EGTA and .2 mM PMSF, because these protease inhibitors improved specific binding by approximately 50% to greater than 150 fmol/mg of protein and decreased non-specific binding to less than 10% at 2.5 nM DHA.

Leukocyte Migration in Adipose Tissue of Mice Null for ICAM‐1 and Mac‐1 Adhesion Receptors

“…Adipose tissue was minced and incubated in Krebs‐Ringer bicarbonate buffer (3 mL/g adipose) containing 10 mM glucose and 4% bovine serum albumin, incubated with 840 U/g collagenase type I (Worthington Chemicals, Lakewood, NJ) at 37 °C with gentle agitation for 1 hour, and filtered through chiffon mesh into a graduated cylinder (7,8). The lipid‐laden adipocytes floated, and the infranatant fraction was removed and centrifuged at 200 g to pellet the S/V cells.…”