Comet Assay to Quantify DNA Damage in FLT3 Mutant-expressing 32D Cells after Exposure to Type I and Type II FLT3 Inhibitors

We employ the comet assay to systematically evaluate the genotoxicity of type I and type II FLT3 inhibitors on 32D FLT3 mutant cells, providing critical data for clinical optimization in AML. During the procedures, improper handling microsurgery tool will alter the slide, resulting in sample loss. Research indicates that AC220 as the type II inhibitor inflicts more severe DNA damage upon FLT3 mutant cells, providing crucial insights for developing combination therapies by exploiting DNA damage vulnerability to overcome therapeutic resistance.

Investigate the mechanism of action of FLT3 inhibitors in the treatment of AML, and develop dual-drug or multi-drug combination therapy strategies based on FLT3 inhibitors. To begin, prepare a 10 to one mixture of low melting point agarose and cell suspension in 1.5 milliliter tubes using a pipette, and mixed gently. Dispense 55 microliter aliquots of the agarose cell mixture onto comet slides using a pipette held at a 45 degree angle.

Then, place the slides coated with the mixture at four degrees Celsius for 30 minutes in a dark environment. Submerge the solidified slides in pre-cooled lysis solution and incubate for at least one hour at four degrees Celsius, protecting the slides from light. After lysis, remove the slides from the lysis solution.

Using a pipette gun, carefully suck up as much residual liquid as possible from around the paddle. Then, place the slides in alkaline electrophoresis buffer and allow them to pre-equilibrate for one hour at four degrees Celsius in the dark. Now, place the slides into the electrophoresis chamber.

To fully immerse the slides, add enough pre-cooled alkaline electrophoresis solution. Run electrophoresis at 24 volts with a constant current for 30 minutes using a buffer pre-equilibrated to reduce DNA damage. After electrophoresis, use a pipette gun to aspirate the alkaline electrophoresis buffer from around the slide.

Submerge the slides in double distilled water for five minutes, then submerge the slides in 70%ethanol for five minutes. Place the slides in an incubator set at 37 degrees Celsius for 15 minutes to dry. Next, apply a 100 microliter aliquot of YeaRed nucleic acid stain to each well of the slide.

Incubate the slides for 30 minutes at 28 degrees Celsius in a light-protected environment. Rinse the slides gently with water to remove excess stain and dry them in a 37 degrees Celsius incubator. The comet assay was systematically employed to quantify differential DNA damage profiles induced by Gilteritinib and AC220 in FLT3 mutant cell lines.

Quantitative analysis of tail DNA percentage confirmed that both compounds significantly increase DNA damage after two, four, and six hours, with AC220 producing higher damage levels than Gilteritinib at each time point. All of moment tail measurements mirrored the tail DNA trend with statistically significant increases for both treatments from two to six hours and higher OTM values observed in the AC220 group throughout.