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- ItemRaman spectroscopy follows time-dependent changes in T lymphocytes isolated from spleen of endotoxemic mice(Rockville : American Association of Immunologists, 2019) Ramoji, Anuradha; Ryabchykov, Oleg; Galler, Kerstin; Tannert, Astrid; Markwart, Robby; Requardt, Robert Pascal; Rubio, Ignacio; Bauer, Michael; Bocklitz, Thomas W.; Popp, Jürgen; Neugebauer, UteT lymphocytes (T cells) are highly specialized members of the adaptive immune system and hold the key to the understanding the hosts’ response toward invading pathogen or pathogen-associated molecular patterns such as LPS. In this study, noninvasive Raman spectroscopy is presented as a label-free method to follow LPS-induced changes in splenic T cells during acute and postacute inflammatory phases (1, 4, 10, and 30 d) with a special focus on CD4+ and CD8+ T cells of endotoxemic C57BL/6 mice. Raman spectral analysis reveals highest chemical differences between CD4+ and CD8+ T cells originating from the control and LPS-treated mice during acute inflammation, and the differences are visible up to 10 d after the LPS insult. In the postacute phase, CD4+ and CD8+ T cells from treated and untreated mice could not be differentiated anymore, suggesting that T cells largely regained their original status. In sum, the biological information obtained from Raman spectra agrees with immunological readouts demonstrating that Raman spectroscopy is a well-suited, label-free method for following splenic T cell activation in systemic inflammation from acute to postacute phases. The method can also be applied to directly study tissue sections as is demonstrated for spleen tissue one day after LPS insult.T lymphocytes (T cells) are highly specialized members of the adaptive immune system and hold the key to the understanding the hosts’ response toward invading pathogen or pathogen-associated molecular patterns such as LPS. In this study, noninvasive Raman spectroscopy is presented as a label-free method to follow LPS-induced changes in splenic T cells during acute and postacute inflammatory phases (1, 4, 10, and 30 d) with a special focus on CD4+ and CD8+ T cells of endotoxemic C57BL/6 mice. Raman spectral analysis reveals highest chemical differences between CD4+ and CD8+ T cells originating from the control and LPS-treated mice during acute inflammation, and the differences are visible up to 10 d after the LPS insult. In the postacute phase, CD4+ and CD8+ T cells from treated and untreated mice could not be differentiated anymore, suggesting that T cells largely regained their original status. In sum, the biological information obtained from Raman spectra agrees with immunological readouts demonstrating that Raman spectroscopy is a well-suited, label-free method for following splenic T cell activation in systemic inflammation from acute to postacute phases. The method can also be applied to directly study tissue sections as is demonstrated for spleen tissue one day after LPS insult.
- ItemNew perspectives for viability studies with high-content analysis Raman spectroscopy (HCA-RS)(Berlin : Nature Publishing, 2019) Mondol, Abdullah S.; Töpfer, Natalie; Rüger, Jan; Neugebauer, Ute; Popp, Jürgen; Schie, Iwan W.Raman spectroscopy has been widely used in clinical and molecular biological studies, providing high chemical specificity without the necessity of labels and with little-to-no sample preparation. However, currently performed Raman-based studies of eukaryotic cells are still very laborious and time-consuming, resulting in a low number of sampled cells and questionable statistical validations. Furthermore, the approach requires a trained specialist to perform and analyze the experiments, rendering the method less attractive for most laboratories. In this work, we present a new high-content analysis Raman spectroscopy (HCA-RS) platform that overcomes the current challenges of conventional Raman spectroscopy implementations. HCA-RS allows sampling of a large number of cells under different physiological conditions without any user interaction. The performance of the approach is successfully demonstrated by the development of a Raman-based cell viability assay, i.e., the effect of doxorubicin concentration on monocytic THP-1 cells. A statistical model, principal component analysis combined with support vector machine (PCA-SVM), was found to successfully predict the percentage of viable cells in a mixed population and is in good agreement to results obtained by a standard cell viability assay. This study demonstrates the potential of Raman spectroscopy as a standard high-throughput tool for clinical and biological applications.