Overview Procedure for the CometChip® (Alkaline Assay Protocol)

The Assay Protocol described below is written as a Quick Reference for one CometChip®. Reagents and detailed instructions, including reagent preparation, are provided in the complete instruction guide.

  1. Equilibrate CometChip® in 100 ml PBS at room temperature.

  2. Cool 100 ml Lysis Solution and 1 L Alkaline Solution to 4°C. Melt 5 ml LMAgarose and cool in a 40°C water bath. Cool the electrophoresis tank to 4°C.

  3. Prepare a cell suspension at >1.0 x 10^5 cells/ml. 100 ml will be added to each well. (Standard concentration is 1.0 x 10^6; effective range: 10^5–5x10^6)

  4. Using the purple key, carefully place CometChip® into the 96-Well CometChip® device.

  5. Aliquot 100 µl cells per well, cover with lid and place in tissue culture incubator for 20 minutes. Half way through the incubation period, gently rock E-W and N-S.

  6. Use purple key to carefully remove CometChip® from the 96-Well CometChip® device and gently rinse with 5 ml PBS. Use a microscope to confirm cell loading.

  7. Overlay CometChip® with up to 5 ml LMAgarose. Allow to set for 3 min then transfer to 4°C for 3 minutes to completely solidify.

  8. Place the CometChip® back into the 96-Well CometChip device.

  9. Add treatment solution (media plus chemical at selected concentrations) and incubate in tissue culture incubator. Use three wells per condition, varying either dose or time.

  10. Aspirate media, carefully remove the CometChip® and place the CometChip® 100 ml Lysis Solution for 1-24 hours.

  11. Place CometChip® in electrophoresis tank and submerge in Alkaline Solution. Incubate 60 min.

  12. Electrophorese at 1 V/cm at 4°C.

  13. Neutralize CometChip® for 15 min at 4°C in PBS.

  14. Stain for 20 min at 4°C in 100 ml 1X  SYBR® Gold

  15. Destain 20 min at room temperature in 100 ml PBS. Store at 4°C in the dark until ready to image.

  16. Score wells at 4X using fluorescence microscope with fluorescein filter.

CometChip Protocol Variations:

  • Single time point, multiple doses.

  • Single dose, multiple time points.

  • Pretreat cells off-chip. When treatment ends, load the CometChip®. Optional: Keep cold until lysis to suppress repair.