We have investigated in situ real-time monitoring of apoptosis on human promyelocytic leukemia (HL-60) cells using infrared absorption spectroscopy with the multiple internal reflection (MIR-IRAS) geometry. Actinomycin D (Act D)-induced apoptosis on HL-60 cells was monitored for 24 h. Apoptotic cells showed two strong peaks around the protein amide I and amide II bands probably due to the leakage of cytoplasmic proteins, while growing viable cells showed a peak corresponding to the secretion of metabolites and two downward peaks corresponding to uptake of nutrients from culture media. In addition, IR absorption peak intensity of the amide I and amide II bands was proportional to the extracellular concentration of lactate dehydrogenase, a marker protein for cell damage. These results demonstrate that our MIR-IRAS method is useful for discrimination of apoptotic cells from viable ones and cell apoptotic processes can be monitored in situ by analyzing the amide I and amide II peak intensity.
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