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Why is data C required after data B acquisition?
Why is it required to wait a few hours in step 3?
What is the best sample preparation protocol for pancreatic islet cells?
Asked on Nov 14 2014
Thank you for contacting us. Regarding your questions from yesterday:
1. Why is data C required after data B acquisition?
The two readings should be similar and perhaps data C is not necessary but the ATP signal may go down slightly while the ADP converting enzyme is prepared.
2. Why wait a few hours after adding the ATP Monitoring Enzyme and Nucleotide Releasing Buffer to the wells and before reading data A?
There may be bacterial contamination in the well or reagents that will produce some ATP signal, which should disappear after a few hours. Again, the wait may not be necessary if there is no contamination. (The note in step 3 about waiting a few hours should probably be at the start of step 3. There is no need to wait a few hours AFTER reading data A).
3. What is the best sample preparation protocol for pancreatic islet cells?
I recommend incubating each islet sample with 50ul of the Nucleotide Releasing Buffer, incubating for 5 minutes at room temperature with gentle shaking, and then adding this mixture to the ATP Monitoring Enzyme and Nucleotide Releasing Buffer mixture in the assay plate.
Tom RuyleAbcam Scientific Support
Answered on Nov 14 2014