Fig. 6.

Measurement of intracellular Ca²⁺ in colonic crypt primary cultures. Rats were fed either fish oil (FO, n-3 PUFA) or corn oil (CO, n-6 PUFA)-enriched diets for 3 wk, and colonic crypts were isolated and subsequently incubated with or without 5 mM butyrate (B) for 30 min. Crypts were coin-cubated with fluo-4 AM (3 μM) and rhod-2 AM (2 μM) for 30 min and the mitochondrial-to-cytosolic Ca²⁺ ratio was determined. A: representative fluorescence image of crypts coloaded with fluo-4 and rhod-2. B: example of select cultures incubated with calcein (green) and ethidium homodimer (orange) to assess cell viability. C: relative mitochondrial-to-cytosol Ca²⁺ ratios in isolated crypts under basal conditions, no butyrate added to the culture media. D: effect of exogenous butyrate on mitochondrial Ca²⁺ levels. Data are means ± SE from 15 rats per treatment group with at least 15 images per rat. Bars not sharing a common letter are significantly different, P < 0.001. Bars not sharing a common letter are significantly different at P < 0.05.