Stem Cell Assays

Overview of GPCR stimulation assay

Upon reaching subconfluency, BMSCs are stimulated with specific GPCR agonists (blue arrow) and the cellular response was detected by the xCELLigence system. (B) A close look at the stimulation response observed in (A) (dotted box). Cell index was normalized against the cell index at the time of treatment (NCI). The response window is calculated as the maximum NCI (NCImax) reached upon treatment for a given time point, divided by the NCI of the negative control (NCInegcntl) at that time point.

Application Note: Functional​​​​​​ Assessment of Mesenchymal Stem Cells

Distinct GPCR Profiles in Undifferentiated versus Differentiated BMSCs Provides a Rapid Prediction of Functional Status.

BMSCs from multiple donors were expanded over 12 passages. At selected passages, small cell samples were seeded on E-plates, induced toward adipogenesis, and tracked on xCELLigence to monitor functional capacity in real time.  

GPCR Signatures as Rapid QC Markers

BMSCs exhibit distinct GPCR activity profiles that can be leveraged for quick and reliable quality control. Using the xCELLigence GPCR stimulation assay, you can:

• Assess GPCR responsiveness as a direct indicator of MSC functional integrity
• Incorporate quality checks seamlessly during cell expansion
• Use only a small number of cells (≥1,100 cells/well in 96-well plates)
• Obtain actionable results within just a few days—or as early as 48 hours when using higher cell densities (10–20K cells/well)

Quantitative Correlation

Activation of A1AR, P2Y1, and H1 receptors correlates with adipogenic differentiation capacity. This provides a fast, predictive readout of MSC quality and functional integrity.

• Early-passage cells: high GPCR signaling and strong differentiation potential
• Later passages: reduced receptor activity and declining potency

Quantitative Metrics for Confident MSC Characterization

xCELLigence RTCA growth curves provide several powerful parameters:

• CImax – Maximum Cell Index
• GT50 – Time to reach 50% of CImax
• CIDT – Cell Index Doubling Time during logarithmic growth

These metrics deliver a highly quantitative view of MSC quality and performance.

Ap​​​​​​plication Note: Quality Control and Functional Assessment of Mesenchymal Stem Cells

Quality Control Across Donors and Passages

Using xCELLigence RTCA, BMSCs from different donors were tracked across 12 passages in xeno-free media.

Key finding:

Real-time growth profiles revealed significant differences in proliferation potential between donors, clearly visible at passages 2, 6, and 10.

This demonstrates the platform’s ability to detect functional decline and donor variability early and reliably.

Quantitative Correlation with Differentiation Potential

GPCR activation in response to A1AR, P2Y1, and H1 receptor agonists correlates closely with adipogenic differentiation potential:

• Early-passage cells (high functional capacity) exhibit strong GPCR signaling and robust receptor expression.
• Mid-to-late passage cells show diminished GPCR activation as they approach senescence, paralleling their decline in differentiation potential.

This measurable correlation provides a rapid, predictive, and quantitative method to assess MSC potency long before traditional endpoint assays can.

Real-time analysis of BMSCs from donors 2 and 3 shows clear functional decline with increasing passage number. Early vs. late passage growth curves (A, G) reveal reduced proliferative capacity over time. Quantitative metrics, including Cell Index Doubling (B, H) and phenotypic marker profiles (E, K), highlight when cells cross predefined thresholds indicating loss of function.

Monitoring Myoblast Differentiation

Human primary satellite cell–derived myoblasts were seeded in E-Plate L8 (20,000 cells/well). After switching to low-serum differentiation medium (50 hours post-seeding), xCELLigence continuously tracked the fusion of myoblasts and formation of mature myotubes.

Figures courtesy of Dr. Andrea Domenighetti, Northwestern University.