DOI: 10.5281/zenodo.7673199
title: In vitro genotoxicity testing using γH2AX biomarker, Microscopy Dataset

description: The Dataset was used and is supplementary to the paper "In vitro genotoxicity testing using γH2AX biomarker, microscopy and automatic image analysis in ImageJ - a pilot study with valinomycin". It contains both RAW single-channel images and numerical results obtained with BioImage Analysis and evaluation available through the GitHub repository: https://github.com/martinschatz-cz/genotoxicity-bia.


Naming Convention for images
ChannelName_YYYYMMD_Well_PossitionInWell_AcqRun.tiff


Naming Convention for results
Well_AllResults_YYYY-MM-DD_Results.csv


The csv are ‘,’ separated, and automatically named by analysis script.



 


Folder Structure



	

	
Images (1069 files, as Images.zip)

	

		

		
4H - all images for all wells
		
		

		
24H - all images for all wells
		
	
	
	

	
Results (36 files)

	

		

		
Results_4H (as Results_4h.zip)
		
		

		
Results_24H (as Results_24h.zip)
		
	
	



 


Measurement Settings



	

	
Manufacturer and model of microscope: Olympus IX83 P2ZF
	
	

	
Objective lens magnification, NA: 10x Olympus IX3 Nosepiece, LensNA=0.3
	
	

	
Excitation filters (mounted in the light source)

	

		

		
Violet:  395/25nm           LED module 1, DAPI
		
		

		
Green: 555/28nm         LED module 5, Cy3
		
	
	
	

	
Quad band filter set for DAPI/FITC/Cy3/Cy5
	
	

	
Quad band polychroic mirror (mounted in the filter turret):

	

		

		
BP 411-454nm,
		
		

		
BP 495-536nm,
		
		

		
BP 577-617nm
		
		

		
BP 655-810nm
		
	
	
	

	
Emission filters (mounted in the fast emission filter wheel, infront the camera):

	

		

		
DAPI:               BP 421-445nm
		
		

		
Cy3:                 BP 581-619nm
		
	
	
	

	
Illumination light source: Lumencor Lumencor Spectra X Lamp
	
	

	
Pixel size: 650nm x 650nm
	
	

	
Camera manufacturer and model: Hamamatsu ORCA-Flash4.0
	
	

	
Software program(s) and version: OLYMPUS cellSens Dimension 3.2 (Build 23706)
	
	

	
Image acquisition settings 

	

		

		
expposure 500 ms
		
		

		
gain: 0
		
		

		
binning: 4 x 4
		
	
	
	

	
Experiment manager: ZDC + autofocus, two channels: DAPI and Cy3
	



 


Image Processing and Analysis
The data analysis workflow consists of several stages, each of which was executed by a specific script. Firstly, the raw data were manually cleaned and automatically sorted and organized using sort_wells.ijm in FIJI. Secondly, image analysis was performed using Process_WFolder_macro_v1.ijm in FIJI, which processed the image data and extracted the relevant features. Finally, the results were further processed using SF_dataVis_and_statistics_mean_XYh.ipynb in Python (Jupyter Notebook), which generated the final output in the form of a CSV file.


 


In this repository, you can access the resulting CSV file, which contains the final results of our analysis. Additionally, we have provided the scripts used to process the data, which are available on our GitHub repository (LINK). You will find instruction how to create local Jupyter Hub for Python scripts. These scripts are accompanied by a short manual that provides an overview of the data analysis workflow and helps users navigate through the code. By making our scripts available, we hope to facilitate transparency and reproducibility of our research. If you encounter any issues, please report them through the GitHub repository: https://github.com/martinschatz-cz/genotoxicity-bia.


We believe that our work can be useful for other researchers and analysts who are interested in studying similar datasets. We invite you to explore the contents of this repository and use the data and scripts provided here to further your research.


 


Cell lines and culture conditions
Human cervical adenocarcinoma (HeLa) and a Chinese hamster ovary (CHO-K1) cell lines were obtained from American Type Culture Collection (ATCC). The HeLa cells were grown MEM supplemented with 10 % FBS and NEAA. CHO-K1 cells were cultivated with DMEM supplemented with L-proline (final concentration 35 mg/l). The cell incubation took place in a humidified atmosphere of 5% CO2 at 37 °C.


 


Direct measurement of DNA DSBs
The cells were seeded in concentration 0,5 × 105 cells/ml into the 96-well plate (VWR, 10062-900). The cells were rinsed by phosphate buffered saline (PBS;) after 24h incubation, and medium with reduced FBS content (5 %) was added. Valinomycin was dissolved in DMSO and added to cells in two final concentrations (30 and 15 𝞵M). After 4h/24h incubation the visualization was done using and following protocol of HCS DNA Damage Kit. The cells were fixed by 4% paraformaldehyde solution for 15 min at room temperature. The cells were rinsed once by PBS and the permeabilization was performed using Triton® X-100 () solution by incubation for 15 min at room temperature. The wells were rinsed with PBS once and the plate was blocked by 1% BSA blocking solution. After 1 hour incubation at room temperature the blocking solution was removed and 100 𝞵l of pH2AX mouse monoclonal antibody solution (1:1000 in BSA) was pipetted into each well incubated for 1 hour at room temperature. After three times rinsing by PBS the 100 𝞵l of Alexa Fluor® 555 goat anti-mouse IgG (H+L; 1:2000) and Hoechst 33342 (1:6000) solution was incubated for 1 hour at room temperature protected from light. After the incubation the wells were rinsed three times by PBS. The plate was stored with 100 𝞵l in the refrigerator (4 °C) until the image analysis was performed.