Fig. 10

Example of Cell Tracker Red (CTR) being used to identify two different treatment groups of islets. a-c Images of islets: group 1 contains control islets loaded with Fura-2 for 30 min, and group 2 contains “test” islets loaded with Fura-2 and 200 nM CTR for 30 min. Both groups of islets are combined in the recording chamber for live-cell fluorescence microscopy. a A brightfield image shows all islets (group 1 and group 2). b An image taken with filters to detect only red fluorescence light (535 nm Ex; 610 nm Em). Test islets (group 2) produce detectable red fluorescence due to the CTR label. Yellow circles surround the location of “invisible islets” from group 1 that were not labeled with CTR. c Fluorescence from the Fura-2 signal (380 nm Ex) is detectable in all islets, so that calcium recordings of group 1 and group 2 can be made simultaneously under identical experimental conditions. d-f An example of a calcium recording using CTR to compare diabetic and healthy islets. d Calcium traces from diabetic islets isolated from db/db mice loaded with 1uM Fura-2 + 200 nM CTR for 30 min. e Calcium traces from healthy islets isolated from heterozygous control mice loaded with 1uM Fura-2 only. f Mean calcium traces for db/db (N=5 islets) and heterozygous controls (N=5) can be used to assess differences in amplitude, slope, and latency by directly comparing two treatment groups under identical environmental conditions