Development of FT-IR microspectroscopy for pre-clinical and clinical applications  (2008)

Bellisola G.; Bergamini G. ; Bolomini-Vittori M.; Cestelli Guidi M.; Cinque G.; Della Peruta M. ; Giagulli C.; Laudanna C.; Mafficini A.; Marcelli A.; Melotti P.; Piccinini M.; Sorio C.
Development of FT-IR microspectroscopy for pre-clinical and clinical applications
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Contributo in atti di convegno
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Contributo in Atti di convegno
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IV meeting DASIM (Diagnostic Applications of synchrotron Infrared Microspectroscopy)
Dublin (Ireland)
12-13 giugno 2008
Fourier Transform Infrared spectroscopy, chronic myelogenous leukaemia, PTPRG
Short description of contents:
The development of new drugs requires high throughput and cost-effective screening infrastructures associated to suitable cellular models1. Fourier Transform Infrared (FT-IR) spectroscopy and microscopy has the potential to detect small and early biochemical changes associated with cellular events, and in combination with pattern recognition/classification methods, it should be utilized as a complementary technique to perform high throughput measurements and screening in pre-clinical drug testing and for routine clinical diagnostic and prognostic analyses2. We explore the possibility to apply FT-IR microspectroscopy to characterize spectral signatures of activation and/or inhibition of intracellular signaling pathways in polymorphonuclear neutrophils (PMNs) incubated ex vivo with specific agonists and/or inhibitors of intracellular phosphonetworks3, a pre-requisite to develop new anti-inflammatory drugs, and to screen the efficacy of different drug formulations in inhibiting a key metabolic pathway relevant for the cell transformation process or to point out which is the most effective in relation to the considered cell model before performing clinical trials. To this scope two distinct clones of K562 cells4, a cell line model of human chronic myelogenous leukaemia (CML), respectively with low and rescued expression of receptor-type protein tyrosin phosphatase gamma (PTPRG) were incubated for 10 minutes with a selective inhibitor of BCR-ABL tyrosine kinase phosphorylation, or with a potent and selective inhibitor of the Src-family tyrosine kinase, respectively. In other experiements, the cystic fibrosis transmittance regulator (CFTR) deficient IB3-1 epithelial cell line and the corresponding isogenic CFTR rescued C38 cell line were incubated with the pro-inflammatory cytokine interleukin 8 (IL-8) or with the antibiotic azitromycin (AZT). At the end of perturbation, cells were fixed in 1% paraformaldehyde buffered solution in PBS, washed with distilled water and cell density was normalized to 5x106 cells/mL. A drop (10 µL) of each sample replicate was deposited on a 2 mm thick ZnSe infrared transparent window. Samples were dried in air and then stored in the vacuum and in the dark until the IR measurements. The acquisition of IR spectra was carried out at DA_NE light laboratory5 (LNF-INFN  Italy) using a Globar source and a Bruker Equinox55 FT-IR interferometer with a KBr beam splitter, connected to a Bruker Hyperion 3000 FT-IR microscope (Bruker Optik GmbH, Ettlingen, Germany) equipped with both 15x magnification objective and condenser. All measurements were performed in air and spectra were acquired in transmission modality from homogeneous zones of the sample containing a comparable number of cells in each selected sample area (typically from a 100x100 µm2 illuminated area, sometime reduced to 20x20 µm2 to obtain the spectrum of single cell). The transmitted IR light was collected by a single element 250x250 µm2 mercury-cadmium-telluride (MCT) liquid nitrogen-cooled detector for both the background and the sample. To obtain an acceptable S/N, 128 or 256 scans were averaged in the 7004000 cm-1 spectral interval with a spectral resolution of 4 cm-1. Absorbance spectra were finally obtained by background spectrum subtraction from each sample spectrum. All spectral manipulations and calculations were performed with the use of OPUS" 6.0 software. Atmosphere compensation was applied. The mean spectrum of replicate measurements of the different sample areas was calculated in the 4000800 cm-1 frequency interval and the 1800800 cm-1 spectral interval was selected and baseline corrected (Rubberband method with 64 points). To identify contribution in peaks, the second-order derivative was computed by using a generalized Savitzky-Golay algorithm with 9 smoothing points. Preliminary results that will be presented and discussed in the poster clearly indicate that FT-IR spectrosc
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Deposited On:
April 9, 2010
Last Modified:
November 2, 2016
Bibliographic citation:
Bellisola G.; Bergamini G. ; Bolomini-Vittori M.; Cestelli Guidi M.; Cinque G.; Della Peruta M. ; Giagulli C.; Laudanna C.; Mafficini A.; Marcelli A.; Melotti P.; Piccinini M.; Sorio C., Development of FT-IR microspectroscopy for pre-clinical and clinical applicationsProceedings of "IV meeting DASIM (Diagnostic Applications of synchrotron Infrared Microspectroscopy)" , Dublin (Ireland) , 12-13 giugno 2008 , 2008

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