Lysosomal flux assay

Change medium to complete medium + bafilomycin (eg 50nanomolar (nM), 100nanomolar (nM), 200nanomolar (nM)).

Treat control wells with complete medium + DMSO (eg 500X, to match bafilomycin dilution) (bafilomycin aliquots are resuspended in DMSO).

Keep at 37°C until designated collection timepoint(s) (e.g. 12 h).

Collect cell pellet.

Keep samples On ice throughout extraction.

Dilute 4X blue LDS buffer (Cat. no. B0007, Life Tech) in water plus protease inhibitors to get 1X LDS buffer.

Resuspend each pellet in 1X LDS buffer (100µL, but reduce or increase the volume according to the pellet size).

Sonicate twice for 0h 0m 15s at 50% power, keeping sample On ice.

Boil for 0h 5m 0s at 100°C; return directly to ice.

Centrifuge for 0h 10m 0s at 850x g,0h 0m 0s.

Retain the supernatant.

Perform BCA assay on protein samples; dilute the standards and the blank in 1X LDS buffer.

Store lysates at -80°C.

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

Boil samples at 100°C for 0h 5m 0s.

Load 25µL per well into 10-well mini Bis-Tris 4-12% gel(s).

Dilute protein ladder in 1X LDS (e.g. 4µL protein ladder + 16µL 1X LDS buffer).

Run in MES buffer (1X), 0h 30m 0s, at 200V.

Cut off wells and bottom of gel; move gel directly to transfer stack.

Transfer using iBlot P0 program to nitrocellulose membrane (Cat. no. IB23002, Thermo).

Do not touch membrane with gloves-use forceps and razor.

Cut off edges (perimeter of gel).

Cut across sample section between 2nd and 3rd (100kDa) ladder bands from top (or somewhere else above 75 KDa).

Cut across samples at 25kDa (lower red) band (middle of the band).

Rehydrate membrane in PBS for 0h 5m 0s on orbital shaker.

Block 1h 0m 0s at100Room temperature in Licor Odyssey Buffer PBS (Cat. no. 927-40000, Licor).

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.

Recover blocking solution to be used for secondary antibodies—keep 4On ice or at 4°C.

Incubate in primary antibody solutions for 2h 0m 0s at 4Room temperature on orbital shaker or at 4°C 1h 0m 0s.

Wash 4x 0h 5m 0s with PBS-T (0.05% Tween).

Incubate secondary antibodies at 1:10,000 dilution in Licor Odyssey plus 0.1% Tween, 1h 0m 0s at 4Room temperature in black box or aluminium foil:

• HMW: 680-anti-rabbit, 800-anti-mouse.
• LMW: 800-anti-rabbit.

Wash 4x 0h 5m 0s with PBS-T (0.05%), in black box or aluminium foil.

Wash 1x 0h 5m 0s with PBS.

Change to fresh PBS.

Immediately before acquisition, dry membranes on kimwipe.

Reassemble membrane and scan with Licor Clx scanner.

• Flip membranes so that lower left corner is in upper left.

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.