## 3 Methodology
### Experimental Studies  
NextGEM aims to collect robust and reliable data by combining different experimental approaches: in vitro studies on human cell lines, in vivo studies on the model organism Caenorhabditis elegans (_C. elegans_), and ex vivo exposure studies on human cells (lymphocytes). Certain in vitro studies will be carried out at more than one lab for replication purposes. Cell models used for experimental studies have been chosen because of the existence of standardized and widely recognized assays (buccal epithelium cells, lymphocytes, neuroblastoma cells), appropriateness for studies of the poorly penetrating 5G signals (keratinocytes), and relative simplicity in the organization and lack of morphological variation (RBC), as can be seen in Figure 3. The experimental studies in NextGEM focus on cancer-related endpoints, reproduction and development, and finally on different biophysical and biochemical mechanisms, as will be described below. All experiments are going to be performed taking into consideration environmental factors such as noise, vibrations, and background magnetic fields and will eliminate these factors. **In vitro studies on mammalian cell lines:** Human neuroblastoma and keratinocyte cell models will be used to investigate the effect of exposure to 4G and 5G signals, given alone, as multiple frequencies, and combined with other agents, to evaluate cancer-related endpoints. Numerical and experimental dosimetry techniques will be employed to design and characterize new exposure systems and/or optimize existing ones and to calculate the dosimetry parameters (specific absorption rate (SAR), electric field, power density) in the sample. The design of the applicator device will be driven by efficiency and SAR uniformity criteria, ensuring the control of environmental (temperature, humidity, CO\({}_{2}\)) parameters according to the recommendations for good laboratory practice (GLP). For dose homogeneity (numerically evaluated through the variation coefficient, i.e., the ratio between standard deviation and average SAR), different solutions have to be adopted in the sub-mmWave band [31] and in the lower frequency band [32]. Several solutions (waveguides, transverse EM cell, radial transmission line, wire patch cell, reverberating chambers, etc.) will be considered. Short (1 to 24 h) and long (3 weeks) exposure durations will be applied, according to the biological systems used and the endpoints measured, at different SAR values, to investigate the effect of 4G (1950 MHz) and 5G (3.5-26.5 GHz) signals. Oxidative stress, apoptosis, and cell cycle progression will be analyzed (flow cytometry,molecular biology techniques). Genomic instability will be evaluated by micronucleleus (MN) and Comet assays. Transcriptomics and epigenetic tests will be performed on selected conditions based on the outcomes of the above-mentioned tests. Two conditions will be investigated on human neuroblastoma cells (apoptosis, cell cycle progression) to test multiple RF-EMF signals: (a) 1800 MHz GSM, 1950 MHz UMTS, and 2.4 GHz WiFi signals and (b) 3.5 GHz and 2.4 GHz WiFi. To address the effect of combined exposures, 4G exposure will be performed in combination with menadione (human neuroblastoma cells) and 5G exposure in combination with UV-B light (human keratinocytes). **In vivo studies on _C. elegans_:**_C. elegans_ will be employed as an in vivo simple model to evaluate cancer-related endpoints and perform genetic screening. Furthermore, reproduction in the parental and second generation will be also assayed. The exposure system is similar to the one adopted for in vitro studies. Oxidative stress and genetic screening will be evaluated on wild-type _C. elegans_ (N2) in vitro with fluorescent markers. For reproduction assays, synchronized nematodes will be exposed at different developmental stages--eggs and L1 to L4 larval stages. Growth, egg and organism development, and motility parameters will be assessed by imaging processing. Gene expression will be assayed as in in vitro and ex vivo experiments.