TITLE: Lysosomal flux assay
AUTHORS: Giacomo Monzio Compagnoni

[DESCRIPTION]
This protocol details the assessment of the autophagic flux in cells, by evaluating LC3II and p62 amount before and after bafilomycin treatment.

[STEPS]
SECTION: Bafilomycin treatment
1. Change medium to complete medium + bafilomycin (eg 50 nanomolar (nM), 100 nanomolar (nM), 200 nanomolar (nM)).

SECTION: Bafilomycin treatment
2. Treat control wells with complete medium + DMSO (eg 500X, to match bafilomycin dilution) (bafilomycin aliquots are resuspended in DMSO).
SECTION: Bafilomycin treatment
3. Keep at 37 °C until designated collection timepoint(s) (e.g. 12 h).

SECTION: Bafilomycin treatment
4. Collect cell pellet.
SECTION: Protein extraction
5. Keep samples on ice throughout extraction.

SECTION: Protein extraction
6. Dilute 4X blue LDS buffer (Cat. no. B0007, Life Tech) in water plus protease inhibitors to get 1X LDS buffer.
SECTION: Protein extraction
7. Resuspend each pellet in 1X LDS buffer (100 µL, but reduce or increase the volume according to the pellet size).

SECTION: Protein extraction
8. Sonicate twice for 15 s at 50% power, keeping sample on ice.

SECTION: Protein extraction
9. Boil for 5 min at 100 °C; return directly to ice.

SECTION: Protein extraction
10. Centrifuge for 10 min at 850 x g.

SECTION: Protein extraction
11. Retain the supernatant.
SECTION: Protein extraction
12. Perform BCA assay on protein samples; dilute the standards and the blank in 1X LDS buffer.
SECTION: Protein extraction
13. Store lysates at -80 °C.

SECTION: Western blot
14. Calculate the volume of each sample containing 30 µg of proteins; add 1X LDS to bring volume to 22.5 µL; add 2.5 µL Thermo Fisher reducing reagent per sample (if using a 10-wells gel).

SECTION: Western blot
15. Boil samples at 100 °C for 5 min.

SECTION: Western blot
16. Load 25 µL per well into 10-well mini Bis-Tris 4-12% gel(s).

SECTION: Western blot
17. Dilute protein ladder in 1X LDS (e.g. 4 µL protein ladder + 16 µL 1X LDS buffer).

SECTION: Western blot
18. Run in MES buffer (1X), 30 min, at 200V.

SECTION: Western blot
19. Cut off wells and bottom of gel; move gel directly to transfer stack.
SECTION: Western blot
20. Transfer using iBlot P0 program to nitrocellulose membrane (Cat. no. IB23002, Thermo).
SECTION: Western blot
21. Do not touch membrane with gloves-use forceps and razor.
SECTION: Western blot
22. Cut off edges (perimeter of gel).
SECTION: Western blot
23. Cut across sample section between 2nd and 3rd (100kDa) ladder bands from top (or somewhere else above 75 KDa).
SECTION: Western blot
24. Cut across samples at 25kDa (lower red) band (middle of the band).
SECTION: Western blot
25. Rehydrate membrane in PBS for 5 min on orbital shaker.

SECTION: Western blot
26. Block 60 min at100 Room temperature in Licor Odyssey Buffer PBS (Cat. no. 927-40000, Licor).

SECTION: Western blot
27. Prepare primary antibody solutions in Odyssey plus 0.1% Tween:

HMW: rabbit-anti-actin 1:1200 (-20 °C) + mouse-anti-p62 1:1000 (labeled “SQSTM1”, at 4 °C).
LMW: rabbit-anti-LC3 1:1000.


SECTION: Western blot
28. Recover blocking solution to be used for secondary antibodies—keep on ice or at 4 °C.

SECTION: Western blot
29. Incubate in primary antibody solutions for 120 min at 4 Room temperature on orbital shaker or at 4 °C 60 min.

SECTION: Western blot
30. Wash 4x 5 min with PBS-T (0.05% Tween).
SECTION: Western blot
31. Incubate secondary antibodies at 1:10,000 dilution in Licor Odyssey plus 0.1% Tween, 60 min at 4 Room temperature in black box or aluminium foil:
HMW: 680-anti-rabbit, 800-anti-mouse.
LMW: 800-anti-rabbit.

SECTION: Western blot
32. Wash 4x 5 min with PBS-T (0.05%), in black box or aluminium foil.

SECTION: Western blot
33. Wash 1x 5 min with PBS.

SECTION: Western blot
34. Change to fresh PBS.
SECTION: Western blot
35. Immediately before acquisition, dry membranes on kimwipe.
SECTION: Western blot
36. Reassemble membrane and scan with Licor Clx scanner.
Flip membranes so that lower left corner is in upper left.
SECTION: Western blot
37. Analysis: LC3 II normalized over Actin (or LC3 I), then divided by baseline (DMSO condition).
LC3 II is larger than LC3 I, but charge makes it run faster: ratio is lower band divided by upper band (or Actin).
Normalize p62 over Actin to corroborate LC3.