Critical pHSense assay parameters

pHSense™ assays can be performed using adherent cells or suspension cells and require no washing step.

As with any assay, it is important to use the correct assay settings when working with pHSense to achieve the desired sensitivity and assay characteristics.

The critical parameters and features to be optimized are listed and briefly commented on below. To get the full details on each parameter optimization, please refer to the manuals of the pHSense reagents and to the "Guide ADC Binder internalization".

• Cell density: Enough cells need to be engaged in the assay to generate a sufficient level of signal. The appropriate cell density will depend on the expression level of the target (receptor) that will be internalized with the pHSense-labeled agent, so the optimal cell density must be determined empirically. Typical cell densities range from 5,000 to 100,000 cells per well.
• Plate type: It is critical to use the recommended plate type to run the pHSense assay. White plates with a white bottom, such as 96-well white culture plates (Revvity, Part #6055680) for adherent cells assays or 384-well white culture plates (Revvity, Part #6007680) for suspension cells assays, are recommended for optimal sensitivity. Clear bottom or black plates are not recommended, as they tend to result in a loss of sensitivity.
• Pre-association: To ensure full labeling of the test antibodies, antibody-labeling reagents (Eu Fab pHSense anti-IgG) should be pre-associated at a high concentration (typically 30 minutes at 200:100 nM) before dilution for testing internalization at lower concentrations.
• Assay medium: pHSense assays can be run in complete culture medium (e.g., DMEM + 10% FBS + antibiotics) or in the taglite labeling medium (Revvity Cat# LABMED). Please refer to the manual of the specific pHSense reagent used to determine the recommended assay buffer for this pHSense reagent. Simple buffers such as PBS or HBSS are NOT recommended for running pHSense experiments, as they can result in an increased level of background signal.
• Medium acidification over time: When cells are highly metabolically active, they may acidify the culture medium after several hours of incubation. This acidification can lead to increased background signal, as the extracellular pHSense dye begins to be activated by the acidic culture medium. Using adequate negative controls will help detect this phenomenon, and the Boost Reagent can minimize its impact. If required, the culture medium can be changed before the reading, or culture media with extra buffering capacity can be used to prevent the medium acidification effect.
• Temperature: As with all pH-sensitive dyes, the pHSense signal is impacted by temperature. To ensure consistent data collection, all plates must be read at the same temperature.
• Kinetic reading: The pHSense dye is resistant to photobleaching. However, some cell types are more sensitive to UV irradiation emitted by certain plate readers. Repetitive readings of the same well can lead to decreased pHSense signals over time due to the UV sensitivity of the cells. This decrease has not been observed with Revvity plates readers (Victor Nivo, EnVision, EnVision Nexus). For other plate readers, caution is advised. Please discuss your setup with your local field application specialist. For more details, please refer to the "Guide ADC binder internalization".
• Negative controls: To demonstrate the specificity of the pHSense signals observed, results are better interpreted by comparison to one or more negative controls. The type of negative control used will depend on the assay setting. For example, for antibody internalization, a pHSense Fab only control and an irrelevant antibody control (see product listing) are useful. For receptor internalization, a no-agonist control serves as the reference. Negative controls help detect non-specific internalization originating from pinocytosis or Fc-receptor mediated internalization. Please refer to the manuals of the pHSense reagents used and the "Guide ADC Binder internalization" for more details on data analysis and interpretation.
• Reader settings: To achieve optimal results, the reader settings must be adapted for the pHSense assay. See the "pHSense Reader Settings" section for practical advice.