Loss of membrane Structure and cytoplasmic volume in glutaraldehydefixed neutrophils compared to cryofixed-freeze-substituted neutrophils

Abstract

Cryofixation instantly preserves cell processes without chemical alteration and freeze-substitution (FS) preserves cell constituents. Investigation has shown that cell membranes and microfilaments appear better preserved in cryofixed-freeze-substituted (CFFS) specimens, including basophils. However, the cells have not been looked at morphometrically nor have neutrophils been looked at specifically.Peripheral blood was collected in heparin from four male donors by venipuncture. Blood was centrifuged at 500 x g for 45 min to separate the granulocytes using a one-step density gradient approach, and then washed in RPMI 1640. Cells were split and half were resuspended in RPMI with 10% DMSO, cryofixed using the Eiko RF-2 and stored in liquid N2 until FS processing in 4% OsO4 in acetone with embedment in Spurr’s resin. The other half were resuspended and fixed in 3% glutaraldehyde in 0.1M Cacodylate, pH 7.35 for 1 hr at 4-6°C. Cells were dehydrated, embedded in Spurr’s resin, counterstained with UALC and examined using a Zeiss EM109.