Summary Dataset of confocal microscopy data of cells exposed to gamma-irradiation and immunostained with gH2AX and 53BP1. Part 1/3: Head and neck primocultures immunostained with gH2AX/53BP1, Testing dataset. DOI 10.5281/zenodo.2564980 Part 2/3: U-87 and NHDF Cells exposed to 1-4 Gy confocal microscopy data of head and neck tumor primocultures immunostained with gH2AX/53BP1. Testing dataset. DOI 10.5281/zenodo.2572450. 174 TIFFs Part 3/3 (this dataset): training dataset for nuclei and gH2AX foci with ground truth annotation masks, head and neck primocultures (head and neck non-tumor and spinocellular tumor cells). DOI 10.5281/zenodo.2576241. 150 TIFFs for nuclei learning incl 150 png nuclei masks + 99 TIFFs for DNA repair foci training Code: the code is available at https://github.com/tomasvicar/LearnFoci Materials and methods Dataset Following cells were used: (1) patient primocultures of patients with spinocellular head and neck tumors (histologically verified tumor and tumor-adjacent tissues), isolation protocol descibed in Svobodova et al, 2017, (2) primary glioblastoma cell line U-87 (ATCC HTB-14, LGC Standards, United Kingdom), (3) primary normal human dermal fibroblasts (NHDF, PromoCell, Hedelberg, Germany) isolated from the dermis of juvenile foreskin or adult skin. The study was conducted in accord with the Helsinki Declaration of 1964 and all subsequent revisions thereof. It was approved by the ethical committee of St. Anne’s Faculty Hospital, Brno. Primoculture cells were cultivated in Pen/Strep antibiotic solution (PAA Laboratories GmbH, Austria) in RPMI-1640 medium with 10% FBS (Biochrom, USA) at 37 °C and 50% CO2 in humidified atmosphere up to 50% confluence. U-87 were grown in Eagle's MEM with 10% FBS. Gamma irradiation The cells were irradiated at the Institute of Biophysics, Czech Academy of Sciences, Brno, Czech Republic in a following schemes: (a) patient-derived primoculture was irradiated with a single dose of 2 Gy (D = 1 Gy/min) of gamma-rays (60Co, Chisostat, Chirana, CR) , (b) U-87 and NHDF cells were irradiated with doses 1, 2, and 4 Gy (D = 1 Gy/min). Cells were irradiated in RPMI 1640 medium (37 °C, normal atmosphere). Confocal microscopy of gammaH2AX and 53BP1 foci immunodetection was consequently performed. Fluorescent staining DNA double strand breaks (DSBs) were quantified in different periods of time post-irradiation (30 min, 8h and 24h post irradiation) by means of $\gamma$H2AX and 53BP1 foci immunodetection combined with confocal microscopy. For details see \cite{falk2007chromatin}. Confocal microscopy The microscopy of samples was performed at the Institute of Biophysics, Czech Academy of Sciences, Brno, Czech Republic. Leica DM RXA microscope (equipped with DMSTC motorized stage, Piezzo z-movement, MicroMax CCD camera, CSU-10 confocal unit and 488, 562, and 714 nm laser diodes with AOTF) was used for acquiring detailed cell images (100× oil immersion Plan Fluotar lens, NA 1.3). Total 50 Z slices was captured with Z step size 0.3 μm. File description all files are compressed hyperstack tiffs (50 Z slices and 3 fluorescent channels, XYCZ order), 100x magnification Confocal_HN_tumor_primocultures_train_nuclei.zip: annotated training subset 1 of dataset of patient-derived primocultures (gamma-irradiated with 2 Gy and controls) used for nuclei segmentation training, 150 FOVs, includes TIFF and manually annotated binary mask for nuclei (data_###.tif and respective mask_###.png) Confocal_HN_tumor_primocultures_train_nuclei_description.xlsx: description of above-mentioned files (tissue type, TNM stage, grade, time post irradiation) Confocal_HN_tumor_primocultures_train_foci.zip: training subset 2 of dataset of patient-derived primocultures (gamma-irradiated with 2 Gy and controls) used for foci identification training, 99 FOVs, includes TIFFs (data_###.tif) Confocal_HN_tumor_primocultures_train_foci_description.xlsx: description of above-mentioned files (tissue type, TNM stage, grade, time post irradiation)