AMP

McLaughlin Lab

7/21/2011

Updated 9/1/11 JEB

ATP Assay Protocol

for

Cells Growing Directly on Culture Vessels (No Coverslips)

ViaLight HS Kit (Lonza, Catalog #LT07-211)

Adapted from Lonza’s Protocol #4: Adherent/suspension cells

Cells grown in Luminescence incompatible plate

Using 96 well format

Luminometer without injectors

Kit Preparation upon initial arrival / first use:

  1. Bring the following reagents to room temperature.
  2. Tris-Acetate Bufferstored at 4oC
  3. ATP Monitoring Reagent (AMR)
  4. Stored at 4oC upon initial receipt
  5. Reconstitute AMR in 10mL of the Tris-Acetate Buffer
  6. Replace cap and mix gently
  7. Allow reagent 15 minutes to equilibrate at room temp
  8. Aliquot unused AMR intoblack, light-protected tubes and store at -20˚C for up to two months

Standard Curve:

  1. ATP Standard
  2. Lonza, Catalog #LT27-008
  3. 0.01M stock solution stored at -20˚C
  4. Remove from -20˚C and allow to thaw
  5. Generate the following dilutions in microcentrifuge tubes

[ATP Molar] / uL of ddH20 / uL of ATP Standard / uL of Previous Dilution to add
10-3 / ------/ 100uL / ------
10-4 / 900uL / ------/ 100ul of 10-3
10-5 / 900uL / ------/ 100ul of 10-4
10-6 / 900uL / ------/ 100ul of 10-5
10-7 / 900uL / ------/ 100ul of 10-6

Assay Protocol

  1. Remove NRR from 4˚C and allow to warm to room temperature.
  2. At least 15 minutes
  3. Remove AMR from -20˚C to thaw before use (Keep protected from light).
  4. You will need 20uL per each well of your 96 well plate and the aliquots are 1mL total…
  5. Warm wash media to room temperature:
  6. Typically 1X PBS
  7. Or some other phenol red free media
  8. Remove culture plate(s) from the incubator and allow to cool to room temperature for at least 5 minutes
  9. Program the plate readeras follows:
  10. (Do so ahead of time because it will need time to calibrate)
  11. Measurement Mode: Luminescence
  12. Integration time: 1000ms
  13. Gain: 150
  14. Plate definition file: COS96ft.pdf
  15. Aspirate culture media from plate and addwash media as follows:
  16. 24 well plate = add 2mL of wash media
  17. 6 well plate = add 3mL of wash media
  18. Aspirate first wash and add fresh wash media as follows:
  19. 24 well plate = add 500uL
  20. 6 well plate = add 1mL
  21. Add appropriate amount of room temperature NRR directly to remaining wash media:
  22. 24 well plate with 500uL of wash media = add 100uL of NRR per well
  23. Total volume = 600uL
  24. 6 well plate with 1mL of wash media = add 300uL of NRR per well
  25. Total volume = 1300uL
  26. Incubate cells in wash media/NRR for10 minutes at room temperature.
  27. Transfer 180uL of cell suspension to a white, 96 well platewith a flat, transparent bottom.
  28. Do this in triplicate from each well
  29. Set plate with remaining volume on ice until later – step #16
  30. Add 100uL of each ATP standard in triplicate to the same 96 well plate
  31. To each ATP standard well add 100uL of NRR
  32. Take the following to an area close to the plate reader:
  33. The entire plate with samples and ATP standard curve loaded
  34. The thawed and light protected AMR
  35. A repeator capable of pipetting 20uL
  36. Add 20uL of AMR to both the ATP standard wells and sample wells using the repeator.
  37. Let sit at room temperature for 2 minutes then immediately read on plate reader
  38. After ATP assay is complete, collect lysed cells from step 10b into appropriately labeled tubes (1 per condition) using a rubber policeman and sonicate.
  39. If using 6 well plate, you can combine 500uL of lysate with 500uL Laemmli + BME and boil 10 minutes to have western samples.
  40. There won’t be enough in 24 well plates.
  41. At this point you can freeze the protein lysates at -20˚C or continue with a protein assay…
  42. Note: You will have to prepare the following to use as a true blank and as the media in which your BSA standards are prepared:
  43. For 24 well samples:
  44. 500uL of PBS
  45. 100uL of NNR
  46. For 6 well samples:
  47. 1mL of PBS
  48. 300uL of NNR