Cell-specific CRCs in co-cultures with Brilliant Sodium

Harnessing Brilliant Sodium and automated imaging, we pinpoint ouabain’s potency on Na⁺/K⁺-ATPase in HEK and CHO cell co-cultures, unveiling drug effects with precision.

By combining Brilliant Sodium with automated image analysis, we calculate cell-specific potencies of ouabain, a Na⁺/K⁺-ATPase inhibitor, in a mixed co-culture of human embryonic kidney (HEK) and Chinese hamster ovarian (CHO) cells. Power your research with our Snapshot-compatible solutions – built for a more complex culture future.

Cell staining and experimental setup

HEK cells were stained with Cytotracker red prior to mixing with CHO cells for plating in a 96 well plate. The following day, cells were loaded with ING-2 AM using our Brilliant Sodium protocol for 1 h. Images were acquired at 5 minute intervals after the addition of varying ouabain concentrations using both GFP (for ING-2) and Texas Red (for Cytotracker red) filter cubes.

Ouabain is an inhibitor of the sodium/potassium-pump, which uses ATP to transport sodium and potassium against their concentration gradient to maintain a cell’s resting potential. When the pump is blocked, an increase in intracellular sodium is observed that can be measured using ING-2, a fluorescent sodium indicator. Different isoforms of Na⁺/K⁺-ATPase are known to have sensitivities to ouabain.

Timelapse videos of cells treated with 167 μM Ouabain (separate channels)

All cell identification

All cells were identified as objects if they fit the following parameters in the GFP channel: Area > 15 um – 100 um, Intensity > 3000 RFUs.

Timelapse video of GFP channel with object overlay

HEK293 cell identification

HEK cells were identified in each frame of the video if they fit the following additional parameters: Texas Red intensity > 5000 RFUs. Objects identified as HEK cells are outlined below. These objects are then overlaid onto aligned GFP images for the analysis. The remainder of the cells captured in the GFP mask are qualified as CHO cells.

Timelapse videos of HEK cell object identification

Data processing

Mean cell fluorescence for each cell population is tracked over time. All data is then normalized to the mean cell fluorescence for that population from the first image, acquired just after the addition of ouabain.

Generating cell-specific concentration response curves

Normalized data points at 45 minutes are used to generate a dose response curve for each cell population. A non-linear fit is used to calculate the IC50 for each population. Unique potencies are identified for human and rodent Na⁺/K⁺-ATPase isoforms that aligns with published data.

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