Home › Cell Biology › Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry
Cell Biology JoVE (Open Access) Citable · DOI

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

DOI: 10.3791/2429-v
What you'll learn
  • ✓ Perform flow cytometry-based quantification of GLUT4 translocation
  • ✓ Prepare and stain cells to detect surface GLUT4 receptor expression
  • ✓ Analyze insulin-stimulated glucose transporter trafficking in myoblasts
  • ✓ Compare GLUT4 translocation between healthy and insulin-resistant cells
Protocol

This protocol describes a rapid technique to quantify the translocation of GLUT4 from the cytoplasm to the plasma membrane of cells by flow cytometry.

Difficulty
intermediate
Total time
~2–3 hours per sample batch (including cell preparation, staining, and flow acquisition)
Model organism
Human myoblasts (primary or cultured cell lines)
Biosafety
BSL-1

Steps

1
Prepare and stain cells for flow cytometry

Treat cells with insulin or vehicle control, then perform surface staining with fluorescent-conjugated antibodies against GLUT4. This labels translocated GLUT4 at the plasma membrane for quantification.

▶ 01:15
2
Acquire flow cytometry data on stained samples

Run stained cell samples through the flow cytometer to collect fluorescence intensity measurements. Establish appropriate voltage and gating parameters to quantify surface GLUT4 levels.

▶ 04:05
3
Analyze GLUT4 upregulation in response to insulin

Compare flow cytometry data between insulin-stimulated and control conditions in myoblasts from healthy versus insulin-resistant donors. Quantify the fold-change in surface GLUT4 to assess translocation efficiency.

▶ 04:39
💬 Comments coming soon