Mechanical damage to murine neuronal-enriched cultures during harvesting: effects on free fatty acids, diglycerides, Na+,K+-ATPase, and lipid peroxidation

Abstract

The most commonly used procedure to harvest cultured cells from petri dishes is to scrape the cells off the plates with a rubber or Teflon policeman. However, the results reported herein demonstrate that this technique, with its associated mechanical trauma, significantly perturbed cell membranes in neuronal-enriched cultures derived from the ventral half of fetal murine spinal cords. This is evidenced by liberation of free fatty acids and diglycerides, partial inhibition of Na+,K+-ATPase activity, and increased malondialdehyde production. Harvesting the cells by freezing, either on liquid nitrogen or dry ice, significantly attenuated these effects. This important observation indicates that mechanical manipulation of cultured cells during harvesting significantly affects subsequent biochemical analyses, particularly those associated with the cell membrane (e.g., membrane lipid metabolism and assay of intrinsic membrane enzymes).