Studying mucin secretion from human bronchial epithelial cell primary cultures

Abstract

Mucin secretion is regulated by extracellular signaling molecules emanating from local, neuronal, or endocrine sources. Quantifying the rate of this secretion is important to understanding how the exocytic process is regulated, and also how goblet/mucous cells synthesize and release mucins under control and pathological conditions. Consequently, measuring mucins in a quantitatively accurate manner is the key to many experiments addressing these issues. This paper describes procedures used to determine agonist-induced mucin secretion from goblet cells in human bronchial epithelial (HBE) cell cultures. It begins with primary epithelial cell culture, offers methods for purifying MUC5AC and MUC5B mucins for standards, and describes five different microtiter plate binding assays which use various probes for mucins. A polymeric mucin-specific antibody is used in standard and sandwich ELISA formats for two assays while the others target the extensive glycosylated domains of mucins with lectin, periodate oxidation, and antibody-based probes. Comparing the data derived from the different assays applied to the same set of samples of HBE cell cultures indicates a qualitative agreement between baseline and agonist stimulated mucin release; however, the polymeric mucin-specific assays yield substantially lower values than the assays using non-specific molecular reporters. These results indicate that the more nonspecific assays are suitable to assess overall secretory responses by goblet cells, but are likely unsuited for specific measurements of polymeric mucins, per se.

Fig. 1

Mucins recovered from HBE cell cultures 12-mm inserts) during a careful wash procedure, and the subsequent baseline and agonist (ATPγS, 100 µM) secretion periods (mean ± SE, n = 6). The conventional mucin subunit ELISA was used for these assays.

Fig. 2

Mucins sampled following baseline and agonist (ATPγS, 100 µM) periods, as measured, on the same set of samples, by the binding assays described above. SU = mucin subunit antibody, for both the ELISA and sandwich (ELISA) assays indicated. Results expressed as the mean ± SE, n = 3; fold increase = agonist stimulated/baseline.

Fig. 3

Foam pad, top piece, to hold 6- or 12-well cluster plates during HBE cell culture wash procedures. The center cutout should hold the plate quite firmly, i.e., it should be necessary to work the plate into the cutout by applying pressure to all sides of the plate as it is inserted. Inset: Image of a cluster plate being inserted into an assembled foam pad.