Alamar Blue (alamarBlue™ assay): A new simple non-radioactive, non-toxic in-vitro assay to monitor corneal proliferation and viability

Purpose. To compare alamar Blue TM Assay with (3H) thymidine uptake in evaluating corneal endothelial viability. Methods. Alamar Blue incorporates a proprietary oxidation-reduction (Redox) indicator that both fluoresces and changes color in response to growth and metabolic activity. Rabbit endothelial cells were cultured up to two months and plated onto microwell plates at concentrations ranging from 1250 to 40,000 cells per well. After 24 hours incubation, 10 uL Alamar Blue was added to each well and absorbance measured at 19, 24, & 48 hr. (3H) thymidine was added to the same wells containing Alamar Blue and radioactivity measured at 14 and 19 hours. Sodium azide was used to create a negative control. Results. At 48 hours, Alamar Blue absorbance was complete at all cell concentrations. At 19 and 24 hours, there was a relationship between mean cell concentration and absorbance. (3H) thymidine uptake on non-confluent cultures demonstrated a linear relationship between cell absorbance and (3H) thymidine incorporation. The simultaneous Alamar Blue assay showed linear absorbance for both confluent and non-confluent cell cultures. Conclusion. In this study Alamar Blue demonstrates several advantages over (3H) thymidine: 1) It is as accurate as (3H)thymidine in assaying proliferation of endothelial cells. 2) As opposed to (3H) thymidine, it can assay non-proliferating endothelial cell metabolism. 3) Allows rapid assessment of large numbers of samples. 4) Is non-toxic to cells or technician. 5) Is non-radioactive. 6) Is less costly. 7) Is non-labor intensive. 8) Allows for continuous monitoring of endothelial cell viability.