Figure 4. Detection of apoptotic human corneal fibroblasts. TUNEL staining was performed after human corneal fibroblasts were co-cultured with Acanthamoebae. TUNEL-positive cells are not detected in cultures of fibroblasts without Acanthamoebae (A). Arrowheads show TUNEL-positive apoptotic corneal fibroblasts cultured with Acanthamoebae (B) and with Actinomysin D (C). Scale bar=100 µm. D: Percentage of Annexin V-positive cells. A significant increase of Annexin V-positive corneal fibroblasts was detected on day 2 or later of culture with Acanthamoebae. More than 50% of corneal fibroblasts were Annexin V-positive on days 4 and 5 (n=4–5). Similar results were obtained with repeated two experiments. NC; Negative control, corneal fibroblasts without Acanthamoebae, Am; Corneal fibroblasts co-cultured with Acanthamoebae. *p<0.05.