Hello, we are trying to standardize comet assay at our lab, however, on the visualization step, we don't observe any comets (or stained DNA nucleoids in the negative control slides). I attach to this post some images of what we're seeing on the microscope, we also tried with dark background but we didn't see comets or stained DNA nucleoids.

For your reference I also include details of the protocol used. Thanks in advance for your help!

Leukocyte isolation. Extraction of whole blood by venipuncture and collection in tubes with anticoagulant (EDTA). After collection of blood, RBC lysis with ammonium chloride Article Collection and storage of human white blood cells for analys...

(we use this protocol with the difference that we mix whole blood/ammonium chloride lysis buffer at 1:3 proportion)(We isolate this way because we don't have access to Ficoll/Lymphoprep/Histopaque). After lysis we resuspend on 500 uL PBS

Treatments. We pellet the cells (centrifugation 300 g, 5 min, 4 C)and resuspend in 1 mL PBS (control) and 1mL (200uM H2O2/PBS solution), then the cells stay with the treatments 30 min at 4 C. After 30 min have passed we pellet the cells as before

Embbeding cells in agarose. We resuspend the pelleted cells with 150 uL LMPA (0.8%, Kept at 37 C on thermoblock) then apply to the slides, cover with coverslip and let solidify at 4 C for 5 min, then take off the coverslips and go to lysis

Lysis. (2.5 M NaCl, 0.1 M EDTA, 10mM Tris, pH 10, Triton X-100 added fresh)(we use the lysis buffer without DMSO, adding dH2O instead), lysis done overnight at 4 C

Unwinding. Slides are put 20 min in electrophoresis chamber with Alkaline electrophoresis buffer (300mM NaOH, 1mM EDTA)

Electrophoresis. After unwinding the power supply is turned on and electrophoresis carried as follows: 0.62 V/cm, 100mA, 32 min.

Neutralization. 3 washes, each one every 5 min, adding dH2O dropwise

Staining. We add 80 uL of staining solution (1:10,000 Gelred/PBS) and let it stain for 15 min, protected from light at room temperature. Afterwards we wash the excess stain with dH2O added dropwise, let dry, put coverslip and go to visualization step.

Visualization. Slides visualization was done using an epifluorescence microscope, the images attached to this post were observed using the 40X objective.