Quantification and Characterization of Colonocytes

A wet preparation of the final cell suspension was viewed under a phase-contrast microscope to check the numbers of cells. Cells were stained with trypan blue (0.4%) to distinguish viable cells from necrotic cells and observed in phase-contrast microscope (PH 2, Nikon, Japan). Cells were also counted in a Coulter Counter (Z2 Coulter Particle Count and Size Analyzer, Beckman Coulter) to obtain counts and size distribution. This SCSR method normally gives the yield of 20–40 million cells per gram of stool and size distribution histograms show the existence of 2 distinct populations, one between 2–5 μ and another between 5–8 μ.[8 (link)9 10 (link),21 (link)] In our experiment in all assays, a total count of 3 × 10³ cells per tube were processed for further staining with fluorochrome-conjugated antibody. To measure the IgA and IgG receptor concentration on viable colonocytes, the cells were incubated with specific antibodies using a standard protocol. In this study, we used IgA-fluorescein isothiocyanate (FITC)-conjugated and IgG-Phycoerythrin (PE)-conjugated antibodies (Sigma) [Figure 1].