Alkaline Comet Assay using the monocytic cell line THP-1 CURRENT

This protocol details the experimental procedure for performing the comet assay, a very sensitive DNA break assay based on single cell gel electrophoresis. The analysis of DNA strand breaks, both single- and double-strand breaks (SSBs and DSBs, respectively), was performed in immune responsive cells. The cell line used was the human monocytic cell line THP-1, an adherent cell type with many known applications in in vitro studies of innate immunity. The comet assay is a robust procedure that allows the accurate and reproducible quantification of DNA damage. Here we describe not only the comet assay step-by-step protocol, but also some important aspects related to troubleshooting.


Introduction
DNA damage, in its many forms, is known to regularly affect cells (1). The toolkit employed by cells to overcome the damage and restore DNA integrity is made of a large collection of sensors and effector molecules and is broadly known as the DNA damage response (DDR, 1). Different types of DNA damage can occur, creating single or double-strand breaks in the DNA molecule (SSB and DSB, respectively). The comet assay has been established long ago for the determination of DNA strand breaks, both SSB and DSB, with the possibility of quantification of the extent of the damage using appropriate sofware packages (reviewed in 2).
Innate immunity is the first line of defense in cells against invading microorganisms and innate responsive cells such as monocytes and the differentiated effector cells, macrophages, are capable of orchestrating responses to fight pathogens (3). Innate imune responses closely associate with DNA damage, both leading to inflammation, but also to beneficial disease tolerance pathways (reviewed in 4).
Here we introduce the alkaline comet assay in the human monocytic cell line THP-1. This type of assay is suitable for the quantification of SSBs and DSBs in a manner that is reproducible, accurate and that can be achieved without the need for complicated equipement and in a time frame that extends for no longer than one week.

Reagents
Cell culture If, on the other hand, the DNA damage is too high compared with previously observed results, make sure the control untreated sample is not already damaged before the treatment. Also, make sure that the agarose was not combined with the cells at a temperature capable of causing DNA damage.

Comet assay
If there are no stained cells when observing at the microscope, confirm without fluorescence that the cells were not lost during any of the steps leading to the electrophoresis or after. If there are samples in the slide but those are not stained, repeat the staining with fresh Sybr Green.

Imaging and counting the comets
If there is too much variability between replicates, increase the number of cells counted and ask another operator to also quantify the damage. If using automated systems, assay the same samples manually.

Time Taken
The times described are an approximation and refers to an assay using only 1 slide; scaling up the number of slides is expected to inclrese the time of the procedure.

Cell culture
Variable, depending on the kinetics of the incubation with the treatments; Usually, 1 day should be enough: cells are plated in the morning, the treatments are performed shortly after and last for up to 6h, after which the cells are collected. 8

Comet assay
3h are expected from the moment the solutions are prepared to the end of the electrophoresis.
Staining of the slide should last 1 h and the stained samples should then be used on the next day only.

Imaging and counting the comets
The time spent at the microscope varies considerably depending on the experience of the user, as it envolves sampling several different fields in the sample, chosing representative fields and taking pictures with as much focus as possible of a large number of cells. So, imaging is expected to take at least 3h, but the researcher may need to collect extra pictures if the initial analysis proves to be inconclusive.
If more pictures are necessary, another session at the microscope may be required a few days after the first session, possibly with the same duration.

Comet score analysis
The time required to analyze the population of cells depends of the type of software used, as some are have more automatic features than others. If done manually, the quantification is very labour intensive and requires many hours in front of the computer. Preferentially, more than one person should quantify the cell population and the results should then be compared.