Do protons and X-rays induce cell-killing in human peripheral blood lymphocytes by different mechanisms?  J. Miszczyk, K. Rawojć, A. Panek, A. Borkowska, P.G.S. Prasanna, M.M. Ahmed, J. Swakoń, A. Gałaś  Clinical and Translational Radiation Oncology  Volume 9, Pages 23-29 (February 2018) DOI: 10.1016/j.ctro.2018.01.004 Copyright © 2018 The Authors Terms and Conditions

Fig. 1 Representative photomicrographs of HPBL after ex vivo irradiation with 3.0 Gy protons following staining with Apoptotic, Necrotic and Healthy Cells Quantification Kit (a, photo: 100× magnification, insert: 250× magnification) compared with representative photos (20× magnification, insert: 60× magnification) used for quantification of control HPBL (0 Gy, b) and HPBL after ex vivo irradiation with 4.0 Gy X-rays (c) and 4.0 Gy protons (d). Presented photos were obtained 1 h post irradiation for donor No. 5. Viable (blue, marked by blue arrows), necrotic (red, marked by red arrows) and apoptotic (green, marked by blue arrows) cells were observed under a fluorescent microscope. (For interpretation of the references to colour in this figure caption, the reader is referred to the web version of this article.) Clinical and Translational Radiation Oncology 2018 9, 23-29DOI: (10.1016/j.ctro.2018.01.004) Copyright © 2018 The Authors Terms and Conditions

Fig. 2 Confirmation of the microscopic observation of apoptosis by the DNA fragmentation assay. Gel-like image showing positive controls. Ladder, Agilent DNA 1000 Kit; Positive Control, Positive control (U937 cells, Apoptotic DNA Ladder Kit); D-0, control for non-irradiated HPBL; D-0.5 through D-4.0, a display of human DNA from 5 donors irradiated with different doses: 0.5, 1.0, 2.0, 3.0 and 4.0-Gy of protons, respectively (Bioanalyzer 2100 Agilent, USA). With increasing doses of radiation there was a qualitative increase in DNA fragmentation, indicative apoptosis as well necrosis, confirming fluorescence microscopy. Clinical and Translational Radiation Oncology 2018 9, 23-29DOI: (10.1016/j.ctro.2018.01.004) Copyright © 2018 The Authors Terms and Conditions

Fig. 3 Percentage of apoptotic (a) and necrotic cells (b) after irradiation with X-rays and protons at 1 h post irradiation at different radiation doses. Cell death was measured using Apoptotic, Necrotic and Healthy Cells Quantification Kit. Error bars represent a standard error of the mean. Clinical and Translational Radiation Oncology 2018 9, 23-29DOI: (10.1016/j.ctro.2018.01.004) Copyright © 2018 The Authors Terms and Conditions

Fig. 4 Percentage of apoptotic (a) and necrotic cells (b) after irradiation with X-rays and protons in lymphocytes 4 h after ex vivo irradiation. Cell death was measured using Apoptotic, Necrotic and Healthy Cells Quantification Kit. Error bars represent a standard error of the mean. Clinical and Translational Radiation Oncology 2018 9, 23-29DOI: (10.1016/j.ctro.2018.01.004) Copyright © 2018 The Authors Terms and Conditions

Fig. 5 Normalized ratios of apoptosis/necrosis for protons and X-rays after 1 and 4-h after irradiation in given dose range. Box plot displays variation within the normalized ratios between apoptotic and necrotic cells. The errors taken into consideration are calculated as approximation errors. Spacing between the different parts of the box indicates the degree of spread in the obtained data, showing outliers. The middle horizontal line within the boxes represents median value of each data set. Clinical and Translational Radiation Oncology 2018 9, 23-29DOI: (10.1016/j.ctro.2018.01.004) Copyright © 2018 The Authors Terms and Conditions