Integrin and transferrin recycling assay

1. Culture cells on coverslips or glass-bottom 35mm cell culture dish to 40-50% confluence.

2. Pre-warm reduced serum media (DMEM w/v 0.5% FBS) to 37°C.

3. Starve cells for 60 minutes at 37°C with reduced serum medium.

4. Label cells with Alexa 488-anti-mouse/rat β1-integrin, Alexa 488-anti-mouse/rat β3-integrin (BioLegend cat# 102212 and 104311, 1:500 dilution), or Alexa 568-transferrin (Thermo Fisher Scientific cat# T23365, 1:500 dilution) in pre-chilled reduced serum medium at 4°C for 30 min.

5. Put culture plates with cells on ice and wash cells with ice-cold PBS quickly (30s) x 2 times and ice-cold medium x 1 time.

6. Remove cells from ice, immediately add pre-warmed medium, and incubate at 37°C for 30 minutes to allow endocytosis.

7. Put cells back on ice after 30 minutes.

8. Quickly wash cells with 0.5% acetic acid, 0.5 M NaCl, pH 3.0, to quench the remaining surface-associated fluorescence labeling. Do not exceed 30s. Immediately wash cells with reduced serum medium x 3 times (30s each).

9. Fix cells with 4% PFA for 15 minutes at room temperature (Recycling time point 0 min) or add pre-warmed full medium (DMEM w/v 10% FBS).

10. Incubate cells at 37°C.

11. To stop recycling, put cells on ice at 15, 30, and 60 minutes after incubation and wash with ice-cold PBS x 3 times. Fix cells with 4% PFA for 15 minutes at room temperature at each time point.

12. Stain cells with Alexa 633-WGA at room temperature for 30 minutes (Wheat Germ Agglutinin, Alexa Fluor™ 633 Conjugate, Thermo Fisher Scientific cat# W21404, 1:300 dilution)

13. Wash with PBS for 3 times. Mount with ProLong Gold antifade mountant (Thermo Fisher Scientific cat# P10144)

14. Proceed for fluorescence microscopy (Representative image in Figure 4-figure supplement 2 C-E and Figure 6B of Qu et.al., 2016 doi: 10.7554/eLife.20417).