Vialight HS Protocol

ViaLight HS Protocol/ATP Assay

Protocol 4: Adherent/suspension cells Luminescence incompatible plate; 96 well format Luminometer without injectors

PREP 1. Bring all reagents up to room temperature before use. a. Reagents – Nucleotide Releasing Agent (NRR) in tissue culture refrigerator, ATP Monitoring Reagent (AMR) aliquots in freezer, Tris- Acetate Buffer 2. Reconstitute the ATP Monitoring Reagent (AMR) in 10mL of Tri-Acetate Buffer 3. Replace the yellow screw cap and mix gently 4. Allow reagent 15 minutes to equilibrate at room temp 5. Aliquot unused reagent into polypropylene tubes and store at -20˚C for up to two months

ASSAY 6. Remove culture plate from the incubator and allow it to cool to room temp for at least 5 minutes 7. At this time also warm up media to wash with - MEM/BSA/Hepes (Use MEM/BSA/Hepes/2xN2 for neurons) 8. Program the plate reader a. Measurement Mode: Luminescence b. Integration time: 1000ms c. Gain: 150 d. Plate definition file: GRE96ft.pdf 9. Set out 6 or 24 well plates (number will vary depending on how many cover slips to transfer and kind of cover slip) and add 300uL of Nucleotide Releasing Agent to each well for large cover slips, 100uL for small cover slips 10. Remove media from culture plate, containing cover slips, with a 25mL pipette 11. Wash cells once with 2-3mL of MEM/BSA/Hepes (Use MEM/BSA/Hepes/2xN2 for neurons) 12. Remove MEM/BSA/Hepes media with a 25mL pipette 13. Transfer cover slips to new well plate with Nucleotide Releasing agent 14. Wait 10 minutes 15. Remove AMR from -20˚C to thaw before use, protect from light 16. Transfer 180uL of cell suspension to a 96 well transparent white plate 17. Take to plate reader and place in holder with plate out of reader 18. Add 100uL of ATP Monitoring Reagent (AMR) to each well using a repeater 19. Let sit RT for 2 minutes then read on plate reader