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In situ real-time monitoring of apoptosis on leukemia cells by surface infrared spectroscopy
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Image of FIG. 1.
FIG. 1.

Procedure for the formation of a short MIR prism. (a) Thermal oxidation of Si wafer, (b) patterning of Si oxide by photolithography, (c) anisotropic etching of Si, and (d) removal of Si oxide. A path for IR light propagation is shown in (d).

Image of FIG. 2.
FIG. 2.

A schematic of the MIR-IRAS measurement system which controls temperature and humidity. A gas mixture containing was humidified by passing through a hot water bottle via a bubbler and introduced to the acrylic chambers through silicone tubes. The temperature of the water trap and the chamber was controlled by thermo controller 1 and 2, respectively.

Image of FIG. 3.
FIG. 3.

Microscopic images of apoptotic [(a) and (b)] and control [(c) and (d)] HL-60 cells after 24 h incubation in the FTIR sample room. [(a) and (c)] Phase contrast images. [(b) and (d)] Fluorescent images after stained with Hoechst 33342. Scale bars: for the phase contrast images and for the fluorescent images. Apoptosis was induced by treating with Act D.

Image of FIG. 4.
FIG. 4.

IR spectral changes of (a) apoptotic and (b) control HL-60 cells during 24 h in the FTIR sample chamber. The reference was the spectrum recorded 30 min after the cells were placed in the FTIR sample room. IR spectral changes for the culture medium during 24 h incubation were subtracted. IRAS spectra of (a) 4000 U/ml LDH and (b) 50 mg/ml DL-lithium lactate and RPMI1640 are also shown. The reference for these spectra was the spectrum of pure water. (b) also shows a difference spectrum of the culture medium containing RPMI1640, FBS, penicillin, and streptomycin before and after incubated with HL-60 cells for 48 h in a incubator. IR spectral changes for the medium during 48 h incubation were subtracted from the raw spectrum.

Image of FIG. 5.
FIG. 5.

Time courses of the peak intensity at (a) (amide II) and (b) (amide I). Typical time courses obtained from one experimental pair of apoptotic and control cells are shown. (c) Time course of LDH concentration in extracellular media. The peak intensity and LDH concentration was normalized to those observed 24 h after apoptosis induction. In the LDH assay experiment, cell suspension was prepared in a same manner as the MIR-IRAS experiment and transferred to a multiwell cell culture plate in a incubator. Cell apoptosis was induced by Act D and extracellular LDH concentration at given time points was determined using LDH-cytotoxic test kit. Each data point represents average of three measurements.

Image of FIG. 6.
FIG. 6.

Statistical correlation between the IR peak intensity at (a) and (b) and the extracellular LDH concentration. Cell death was induced either by Act D or 0.2% Tween20. HL-60 cells were treated with Act D for 10 h (◼, ) or 24 h (●, ), or Tween20 for 5 h (▲, ). Control cells were kept for 5 h (△, ) or 24 h (●, ) without adding any reagents.


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752b84549af89a08dbdd7fdb8b9568b5 journal.articlezxybnytfddd
Scitation: In situ real-time monitoring of apoptosis on leukemia cells by surface infrared spectroscopy