Home › Cell Biology › Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker
Cell Biology JoVE (Open Access) Citable · DOI

Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker

DOI: 10.3791/57153-v
What you'll learn
  • ✓ Induce adipogenic differentiation in human mesenchymal stromal cells
  • ✓ Apply lineage-specific markers to identify mature adipocytes
  • ✓ Quantify differentiation using high-content screening and image analysis
Protocol

Traditional methods of assessing adipogenic differentiation are cheap and easy to use, but are not specific to changes in gene expression. We have developed an assay to quantify mesenchymal cell differentiation into mature adipocytes using a lineage specific marker. This assay has diverse applications across basic research and clinical medicine.

Difficulty
intermediate
Total time
~14–21 days (differentiation protocol ~2 weeks; imaging/analysis ~2–4 hours per sample)
Model organism
Human adipose-derived stromal cells (hADSCs)
Biosafety
BSL-1

Steps

1
Induce differentiation of human adipose stromal cells

Culture and treat human adipose-derived stromal cells with differentiation medium to promote commitment to the adipogenic lineage.

▶ 01:55
2
Stain cells with lineage-specific differentiation markers

Apply immunofluorescence or fluorescent staining using adipocyte-specific markers to identify and visualize differentiated mesenchymal cells.

▶ 03:36
3
Image stained cells using high-content screening

Acquire automated, multi-field microscopy images of stained samples to capture spatial distribution and intensity of marker expression.

▶ 05:53
4
Score and classify individual cells computationally

Use image analysis software to segment cells and assign differentiation scores based on marker expression levels.

▶ 08:23
5
Quantify differentiation using transfluor analysis

Apply transfluor algorithm to measure fluorescence intensity changes and derive quantitative differentiation metrics.

▶ 09:43
6
Interpret representative results and validation

Review sample data outputs, including cell scoring distributions and transfluor quantification, to validate assay performance.

▶ 10:43
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