Question (76855) | Extracellular Oxygen Consumption Assay (ab197243)

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Question

We would like to use this product on skin biopsies (about 8 mm), but the protocol doesn't cover this type of samples. Do you have any experimental data related to tissue samples or protocol tips we can use for the assay?

Answer

You can use a similar set up as the one described in the protocol, starting with the recommended dilution of 1:15 for a 96-wp. However, if tissue samples tested need to be larger, we would suggest that you adjust the assay for use in 48-wp or even 24-wp. In this case, however, you would have to do some asay optimization as it will consume a greater volume of reagents and mineral oil.

For instance, for 48-well plates, the assay volume would increase to 300ul - 400ul, therefore using 2X or more reagent, the assay volume for measurement needs a large enough to yield long enough path length to measure enough reliable signal from the well.

Tissue samples should be prepared in the same way as described in the protocol, but measurement should be conducted in the culture media used to culture the tissues, so the samples can metabolize and consume O2 as normal.

There is no need or benefit of overnigh incubation. The probe is added just prior to kinetic measurement (90-120mins), as it measures the change in Oxygen in the sample over time, which is being consumed by the cells/tissue.

The assay must be optimised for the amount of cells / size of tissue per assay volumes, in 150ul if 96-well plate. Therefore, you should prepare samples of different sizes, place tissue samples in wells and measure kinetically to assess different levels of signal increase, thereby identifying the minimal amount of tissue needed for detectable levels of O2 consumption.

Other recommendations:

Standardise tissue weight and Prep. Use normalisation (ATP, total protein, weighing)
Use precision slice cutter
Store tissue samples in carbogen (95% O2), Pre-equilibrate medium
2-4mm x 200µm samples size can be measured in 48WP under >0.2-0.4ml of mineral Oil. (or similar in 24-well plates)
Pre-warm culture media and instruments at 37°C
Use continuous shaking on the fluorescence plate reader.
Use Top reading

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