Viability in freshly sliced LMS and after 24 h of culture. (A) The viability in freshly sliced LMS was tested using a colorimetric MTS assay to measure cellular metabolic activity in ENDO and EPI LMS. Viable and metabolic active cells reduced yellow MTS to purple formazan. The resulting colored solution was quantified by measuring absorbance at 490 nm. The more absorbance detected at 490 nm, the greater the number of viable and metabolic active cells. Freshly sliced ENDO LMS showed a higher viability compared with freshly sliced EPI LMS (n_endo = 8, n_epi = 8 from four rats; data are compared using Student’s t test). (B) The viability of 24-h cultured LMS was assessed based on their responsiveness to calcium-induced calcium release (CICR). After loading LMS with the fluorescent Ca²⁺ indicator Fluo-8AM, electrical stimulation will induce CICR (visible as a change in fluorescence) in viable cells. Cells not responsive to CICR were assumed to be dead cells. These cells mostly showed up with a very bright fluorescent signal (contoured in red in B), in accordance with Ca²⁺ overload and cell death. On average, 5% of cells on the superior layer of LMS were not responsive to electrical stimulation, both in cultured ENDO and EPI LMS (n_endo = 11, n_epi = 10 from 10 rats; data are compared using Student’s t test).