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.
a. Tris-Acetate Buffer stored at 4oC
b. ATP Monitoring Reagent (AMR)
i. Stored at 4oC upon initial receipt
1. Reconstitute AMR in 10mL of the Tris-Acetate Buffer
2. Replace cap and mix gently
3. Allow reagent 15 minutes to equilibrate at room temp
a. Aliquot unused AMR into black, light-protected tubes and
store at -20˚C for up to two months
Standard Curve:
1. ATP Standard
a. Lonza, Catalog #LT27-008
i. 0.01M stock solution stored at -20˚C
b. Remove from -20˚C and allow to thaw
c. Generate the following dilutions in microcentrifuge tubes
[ATP Molar]
uL of ddH20
uL of ATP Standard
10-3
10-4
10-5
10-6
10-7
-------------900uL
900uL
900uL
900uL
100uL
---------------------------------------------------------------------
uL of Previous
Dilution to add
-----------------100ul of 10-3
100ul of 10-4
100ul of 10-5
100ul of 10-6
Assay Protocol
1. Remove NRR from 4˚C and allow to warm to room temperature.
a. At least 15 minutes
2. Remove AMR from -20˚C to thaw before use (Keep protected from light).
a. You will need 20uL per each well of your 96 well plate and the aliquots are 1mL
total…
3. Warm wash media to room temperature:
a. Typically 1X PBS
i. Or some other phenol red free media
4. Remove culture plate(s) from the incubator and allow to cool to room temperature for at
least 5 minutes
AMP
McLaughlin Lab
7/21/2011
Updated 9/1/11 JEB
5. Program the plate reader as follows:
a. (Do so ahead of time because it will need time to calibrate)
i. Measurement Mode: Luminescence
ii. Integration time: 1000ms
iii. Gain: 150
iv. Plate definition file: COS96ft.pdf
6. Aspirate culture media from plate and add wash media as follows:
a. 24 well plate = add 2mL of wash media
b. 6 well plate = add 3mL of wash media
7. Aspirate first wash and add fresh wash media as follows:
a. 24 well plate = add 500uL
b. 6 well plate = add 1mL
8. Add appropriate amount of room temperature NRR directly to remaining wash media:
a. 24 well plate with 500uL of wash media = add 100uL of NRR per well
i. Total volume = 600uL
b. 6 well plate with 1mL of wash media = add 300uL of NRR per well
i. Total volume = 1300uL
9. Incubate cells in wash media/NRR for 10 minutes at room temperature.
10. Transfer 180uL of cell suspension to a white, 96 well plate with a flat, transparent bottom.
a. Do this in triplicate from each well
b. Set plate with remaining volume on ice until later – step #16
11. Add 100uL of each ATP standard in triplicate to the same 96 well plate
12. To each ATP standard well add 100uL of NRR
13. Take the following to an area close to the plate reader:
a. The entire plate with samples and ATP standard curve loaded
b. The thawed and light protected AMR
c. A repeator capable of pipetting 20uL
14. Add 20uL of AMR to both the ATP standard wells and sample wells using the repeator.
15. Let sit at room temperature for 2 minutes then immediately read on plate reader
16. After ATP assay is complete, collect lysed cells from step 10b into appropriately labeled
tubes (1 per condition) using a rubber policeman and sonicate.
a. If using 6 well plate, you can combine 500uL of lysate with 500uL Laemmli + BME
and boil 10 minutes to have western samples.
i. There won’t be enough in 24 well plates.
b. At this point you can freeze the protein lysates at -20˚C or continue with a protein
assay…
i. 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:
1. For 24 well samples:
a. 500uL of PBS
b. 100uL of NNR
2. For 6 well samples:
a. 1mL of PBS
b. 300uL of NNR