Introduction
As explained in the “pHSense Basics” section, it is important to use the correct reader settings to benefit from the full potential and best sensitivity of the pHSense™ dyes, and pHSense signal must be acquired using a compatible plate reader.
All HTRF-certified readers are compatible with pHSense assays; however, TRF reader settings for pHSense assays differ from standard HTRF settings. For optimal assay performance, a delay time of 1,000 µs and an integration (reading) time of 400 µs are typically required. Using standard HTRF settings would miss the benefit from the lifetime sensitivity to pH and may result in a significantly reduced assay window.
The pHSense assay operates in a single detection mode at 620 nm. If a dual-emission mirror is used, the 665 nm signal should be ignored.
For reader configuration, please refer to the documents below. For other plate readers, please contact your local technical support to obtain appropriate pHSense setup recommendations.
|
BMG |
PHERAstar FS | View document here |
|---|---|---|
| PHERAstar FSX | ||
| PHERAstar PLUS | ||
|
Revvity |
EnVision Nexus | View document here |
| EnVision | ||
| VICTOR Nivo |
The TRF signal evolves over time according to the internalization kinetics of the binder or receptor and the assay conditions (e.g., incubation time, incubation temperature, cell type, culture medium).
Plates can be read multiple times over the course of the incubation period, typically up to 24 hours, without compromising data quality. Please refer to the “Critical pHSense Assay Parameters” section regarding the potential sensitivity to UV illumination of some cell types on some plate readers (also detailed and exemplified in the “Guide ADC binder internalization”.)
For research use only. Not for use in diagnostic procedures.
The information provided above is solely for informational and research purposes only. Revvity assumes no liability or responsibility for any injuries, losses, or damages resulting from the use or misuse of the provided information, and Revvity assumes no liability for any outcomes resulting from the use or misuse of any recommendations. The information is provided on an "as is" basis without warranties of any kind. Users are responsible for determining the suitability of any recommendations for the user's particular research. Any recommendations provided by Revvity should not be considered a substitute for a user's own professional judgment.
