ANF-GFP pulse chase

Protocol for ANF::GFP pulse chase

1. Plate approximately 1–2 × 10⁵ cells per well onto coverslips (Thomas Scientific; #1217N79) placed in 24-well cell culture plates. Add 500 μL per well growth media.
2. Transfect cells with 100 ng ANF::GFP at least 24h after seeding using Lipofectamine 2000 (Thermo Fisher), according to the manufacturer’s instructions.
3. Grow cells for 12–16 h at 37°C in 500 μL growth medium.
4. Incubate cells at 20°C in 500 μL PBS per well for 2 h in a conventional incubator to block the formation of DCVs (pulse).
5. At 30 min before the end of the low temperature block, add 10 μg/ml cyclohexamide to the PBS to block the synthesis of new ANF::GFP.
6. Replace PBS with 500 μL growth medium per well and shift the cells to 37°C for 0, 10, 25, or 50 min (chase).
7. At the end of every time point rinse cells twice with 500 μL PBS per well and fix cells with 500 μL 4% paraformaldehyde (PFA made in PBS) for 20 min at room temperature.
8. Rinse cells twice with 500 μL PBS per well and permeabilize with 500 μL 0.5% Triton X- 100 in PBS for 5 min at room temperature.
9. Rinse cells twice with 500 μL PBS per well and place in 500 μL 5% milk in PBS for 1 h at room temperature.
10. Stain cells with mouse monoclonal anti-GFP (1:300; Santa Cruz; #sc-9996) and rabbit polyclonal anti-TGN38 (1:300; Sigma; #T9826) in 0.5% milk in PBS, at room temperature for 1 h.
11. Wash cells with 500 μL PBS per well three times for 5 min each.
12. Incubate with rhodamine (TRITC) anti-rabbit secondary antibody (1:1000; Jackson Immunoresearch; #111-025-144), and Alexa Fluor 488 anti-mouse secondary antibody (1:1000; Jackson Immunoresearch; #115-545-146) in 0.5% milk in PBS, at room temperature for 1 h.
13. Wash cells with 500 μL PBS per well three times for 5 min each.
14. Mount each cover slip onto glass slides using 20-25 μL Vectashield (Vector Laboratories; H1000) or Prolong Diamond (Life Technologies; P36965).
15. Examine by fluorescence microscopy (e.g. using a Nikon 80i wide-field compound microscope with a 60× oil objective (NA = 1.4)).
16. Score cells in three categories: those that have most of the ANF::GFP concentrated at the TGN (“Golgi-like”), those that have ANF::GFP both at the TGN region and at the cell periphery (“Intermediate”), and those where the ANF::GFP is excluded from the TGN (“Periphery”).
17. Count approximately 50–100 cells per time point.
18. Repeat the experiment three times.