Fig 4. Beta-cell composition of islets after 7 days of tissue culture in control media (—) or media supplemented with 4 different doses of necrostatin-1 (25, 50, 100, or 200 μM of Nec-1) on day 3.

3000 islet equivalents were dissociated into single cells using Accutase, stained with 7-amino-actinomycin D (7-AAD) viability dye and phycoerythrin-conjugated anti-insulin antibody, and analyzed by flow cytometry to quantify the proportion of beta cells in islets. Insulin-positive beta cells were gated on 7-AAD-negative live cells. A) Representative flow cytometry plots of insulin staining. B) Percentage of insulin-positive beta cells. n = 6 for each group. **p < .01. Data are expressed as median and interquartile range.