XF Real-Time ATP Rate Assay well plate set up

Cellular metabolic regulation allows cells to adjust for changes in ATP demand with subsequent changes in ATP production to maintain total intracellular ATP levels. By using the Agilent Seahorse XF Real-Time ATP Rate Assay we can measure total ATP production rates in living cells and to distinguish between the fractions of ATP that are produced from mitochondrial oxidative phosphorylation (OXPHOS) and glycolysis, the two main metabolic pathways responsible for ATP production in mammalian cells. 

The XF Real-Time ATP Rate Assay can also be used to calculate the ideal cell seeding density. By creating four different seeding density sources ie 0.5X, 1.0X, 2.0X and 4.0X we can determine which level for our particular cells is best. Optimal cell seeding numbers vary widely, though are typically between 5 × 10³ – 4 × 10⁴ cells per well and must be determined empirically. (For example, for 1 x 10⁴ cells per well, resuspend cells 1 x 10⁴ per 80 μL = 1.25 x 10⁵ cells per mL)

To simultaneously test the ideal seeding level for both Parkinson's and healthy control cells the following well plate design is proposed.

How many XFe-96 cartridges ?

In the updated XFe-96 cartridge well plate set-up below, we can compare between the age groups and genders between healthy controls and Parkinson's patients for study 1, as well as running a duplicate on the same plate.

This offers the advantage of both initial experiment and confirmation run being done under the same experimental conditions. 

One of the issues yet to be understood is the expectations for the number of runs required due to the variability or accuracy of the XFe-96 results, ie how many technical references are needed for this type of experiment - is one duplicate sufficient or do we need to run another plate to increase the testing from two cases to four cases ?

The good news is that if we need further plates the costs seem to be affordable if we assume we can buy the extra cartridges from the Binger laboratory rather than a bulk order from Agilent

OXPHOX XFe-96 well plate design

The Seahorse XFe-96 well plate designs in the confirmation paper were based on comparing just PD samples across different age groups or gender.  In the design below comparisons with the healthy controls (HC) would have been made difficult because the HC sample were planned to be on different plates ie plates for the PD and plates for the HC.

In the updated well plate design we now have both the PD and HC samples for the same age category or gender in the same well plate. This design is better in that both the PD and HC samples are subject to the same treatments in the same XFe-96 test run rather than running two separate plates.

Under the new design, the samples for HC and PD will be prepared at the same time and submitted to the various inhibitors around the same time in the micro-chambers.