Abstract
Three different methods of enzymatic treatment were compared to determine the optimum method to isolate goblet cells. For each method, tracheas were excised from adult mongrel cats, trimmed of excess tissue and cut into segments. Thermolysin Method (TH): Tracheal rings were agitated in a solution of 60 u/ml Thermolysin in PBS. After each of 5 agitation periods, the cell suspension was centrifuged into a 10-6 M EDTA solution. The cell pellet was washed, pipetted × 10, and separated on a 45% Percoll gradient formed at 20,000 ×g for 25 min. Pronase Method (PR): Tracheal segments were incubated in .5% Pronase (Type B:45,000 PUK/gm) and .1% BSA in media 1-9 for 55 min. The surface epithelium was removed by pipette and gentle scraping. The cell pellet was washed and separated as described above. Elastase Method (EL): Tracheal segments were incubated in 20 mM EDTA in Ca, Mg free EBSS for 1½ hours after which the surface epithelium was removed. The epithelium was incubated in Elastase 40 u/ml in EBSS for 30 min. The cell pellet was then incubated in DNAase 20,000 u/ml for 1 hour and then separated on the Percoll gradient. TH yielded no goblet cells which excluded trypan blue or appeared intact by electron microscopy. PR yielded 2 × 106 cells per trachea of which 10% were goblet cells with excellent viability and intact ultrastructure. EL yielded 3.3 × 105 healthy appearing goblet cells/trachea with virtually no contamination by other tracheal cell types. EL appears to be superior to PR or TH as a method to isolate cat tracheal goblet cells.
Article PDF
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Sherman, J., Carr, T. A COMPARISON OF DIFFERENT METHODS TO ISOLATE GOBLET CELLS. Pediatr Res 18 (Suppl 4), 404 (1984). https://doi.org/10.1203/00006450-198404001-01869
Issue Date:
DOI: https://doi.org/10.1203/00006450-198404001-01869