Jul 12, 2022

Public workspaceLysosomal flux assay

DOI
dx.doi.org/10.17504/protocols.io.q26g786j3lwz/v1
  • Giacomo Monzio Compagnoni1
  • 1Brigham Womens Hospital, Harvard Medical School
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DOI: dx.doi.org/10.17504/protocols.io.q26g786j3lwz/v1
Protocol Citation: Giacomo Monzio Compagnoni 2022. Lysosomal flux assay. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g786j3lwz/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: May 15, 2021
Last Modified: May 31, 2024
Protocol Integer ID: 49979
Keywords: Lysosomal flux assay, Autophagic flux, LC3II, p62, Bafilomycin, ASAPCRN
Abstract
This protocol details the assessment of the autophagic flux in cells, by evaluating LC3II and p62 amount before and after bafilomycin treatment.
Attachments
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Materials
MATERIALS AND REAGENTS:

  • Western blot running tank
  • iBlot transfer system
  • LI-COR Clx scanner
  • bench centrifuge
  • 4-12 % Bis-Tris gel
  • iBlot nitrocellulose stacks
  • LDS 4X buffer
  • Protease/phosphatase inhibitors
  • Bafilomycin
  • PBS
  • Odyssey blocking buffer
  • tween20
  • ReagentLC3B AntibodyCell Signaling TechnologyCatalog #2775
  • ReagentAnti-SQSTM1 Antibody clone 20F1.1Merck MilliporeCatalog #MABN130
  • Actin antibody (Sigma A2066)ReagentAnti-Actin antibody produced in rabbitSigma AldrichCatalog #A2066
  • Secondary antibodies (800 anti Rb, 800 anti Ms, 680 anti Rb)
  • ReagentiBlot™ 2 Transfer Stacks, nitrocellulose, miniThermo FisherCatalog #IB23002
  • ReagentOdyssey® Blocking Buffer (PBS)LicorCatalog #927-40000




Bafilomycin treatment
Bafilomycin treatment
Change medium to complete medium + bafilomycin (eg Concentration50 nanomolar (nM) , Concentration100 nanomolar (nM) , Concentration200 nanomolar (nM) ).

Treat control wells with complete medium + DMSO (eg 500X, to match bafilomycin dilution) (bafilomycin aliquots are resuspended in DMSO).
Keep at Temperature37 °C until designated collection timepoint(s) (e.g. 12 h).

Collect cell pellet.
Protein extraction
Protein extraction
15m 15s
15m 15s
Keep samples TemperatureOn 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 (Amount100 µL , but reduce or increase the volume according to the pellet size).

Sonicate twice for Duration00:00:15 at 50% power, keeping sample TemperatureOn ice .

15s
Boil for Duration00:05:00 at Temperature100 °C ; return directly to ice.

5m
Centrifuge for Duration00:10:00 at Centrifigation850 x g .

10m
Centrifigation
Retain the supernatant.
Perform BCA assay on protein samples; dilute the standards and the blank in 1X LDS buffer.
Store lysates at Temperature-80 °C .

Western blot
Western blot
35m
35m
Calculate the volume of each sample containing Amount30 µg of proteins; add 1X LDS to bring volume to Amount22.5 µL ; add Amount2.5 µL Thermo Fisher reducing reagent per sample (if using a 10-wells gel).

Pipetting
Boil samples at Temperature100 °C for Duration00:05:00 .

5m
Load Amount25 µL per well into 10-well mini Bis-Tris 4-12% gel(s).

Pipetting
Dilute protein ladder in 1X LDS (e.g. Amount4 µL protein ladder + Amount16 µL 1X LDS buffer).

Run in MES buffer (1X), Duration00:30:00 , at 200V.

30m
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 Duration00:05:00 on orbital shaker.

5m
Block Duration01:00:00 atTemperatureRoom temperature in Licor Odyssey Buffer PBS (Cat. no. 927-40000, Licor).

1h
Prepare primary antibody solutions in Odyssey plus 0.1% Tween:

  • HMW: rabbit-anti-actin 1:1200 (Temperature-20 °C ) + mouse-anti-p62 1:1000 (labeled “SQSTM1”, at Temperature4 °C ).
  • LMW: rabbit-anti-LC3 1:1000.


Recover blocking solution to be used for secondary antibodies—keep TemperatureOn ice or at Temperature4 °C .

Incubate in primary antibody solutions for Duration02:00:00 at TemperatureRoom temperature on orbital shaker or at Temperature4 °C DurationOvernight .

3h
Incubation
Overnight
Wash 4x Duration00:05:00 with PBS-T (0.05% Tween).
5m
Wash
Incubate secondary antibodies at 1:10,000 dilution in Licor Odyssey plus 0.1% Tween, Duration01:00:00 at TemperatureRoom temperature in black box or aluminium foil:
  • HMW: 680-anti-rabbit, 800-anti-mouse.
  • LMW: 800-anti-rabbit.

1h
Incubation
Wash 4x Duration00:05:00 with PBS-T (0.05%), in black box or aluminium foil.

5m
Wash
Wash 1x Duration00:05:00 with PBS.

5m
Wash
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.