Apoptosis and necrosis quantification

To quantify apoptosis and necrosis in our ex vivo HPBL model; Apoptotic, Necrotic and Healthy Cells Quantification Kit (Biotium, Inc., Hayward, USA) was used. The kit allows simultaneous quantification of apoptotic, necrotic and healthy cells. Identification and discrimination of apoptotic and necrotic cells in vitro can be challenging, especially late stage apoptosis from necrosis [16]. Biotium kit cannot distinguish late apoptosis from necrosis. We preferred to use fluorescence microscopy with the Biotium kit over flow cytometry for quantitative measurements of apoptosis and necrosis and then used apoptotic ladder kit for confirmation of apoptosis (see descriptions below). In this test, HPBL were washed in PBS and resuspended in 1X binding buffer, then 5 µl of FITC-annexin V, ethidium homodimer III and Hoechst 33,342 solution was added to each tube and incubated for 15 min at 21 °C in dark. HPBL were then washed 2 times with 1X binding buffer, fixed with 2% formamide, placed on a glass slide and covered with a glass coverslip.

Generally, 4–6 representative fields of at least 100 cells per dose, per time point were analyzed separately from 3 independent triplicates (slides), by two independent scorers using fluorescent microscopy coupled to an image analysis system (MetaSystems™, Altlussheim, Germany), according to the criteria described by Zhang et al. [17]. Experiments and irradiations were repeated twice each. All slides were coded, blinded to scorers. Sample decoding was done after completing the microscopic evaluation of all slides used for the study to maintain scientific rigor and quality. The results were not statistically different between the slides, repetitions nor scorers; therefore, results are presented as mean values.