May 11, 2026
GFP-mCherry-LC3B Flux analysis by FACS
Forked from HaloTag autophagy flux assay
- Devin M. Fuller1,2,
- Thomas Melia1,2
- 1Department of Cell Biology, Yale University School of Medicine, New Haven, CT;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, 20 MD
Protocol Citation: Devin M. Fuller, Thomas Melia 2026. GFP-mCherry-LC3B Flux analysis by FACS. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl84ow7g2w/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: April 10, 2026
Last Modified: May 11, 2026
Protocol Integer ID: 314828
Keywords: halotag autophagy flux, quantifying autophagy flux, autophagy flux, lc3b flux analysis by facs fac, lc3b flux analysis, facs fac
Funders Acknowledgements:
National Institutes of Health
Grant ID: R01 GM100930
National Institutes of Health
Grant ID: R35 GM153482
National Institutes of Health
Grant ID: DA018343
National Institutes of Health
Grant ID: F31 AG079606
National Institutes of Health
Grant ID: F31 DK136246
Aligning Science Across Parkinson’s
Grant ID: ASAP-025173
Human Frontier Science Program
Grant ID: LT000056/2020-C
Abstract
FACS based method for quantifying autophagy flux used an endogenous reporter.
Materials
H4 GFP-mCherry-LC3B KI cells : https://doi.org/10.7554/eLife.50034
Cell culture materials:
DMEM (Thermo Fisher Scientific, 11965-092)
FBS (Thermo Fisher Scientific, 16140-071)
Penicillin/Streptomicin (10,000 U/mL; Thermo Fisher Scientific, 15140122)
PBS (Thermo Fisher Scientific, 10010023)
Earle’s Balanced Salt Solution (EBSS; Thermo Fisher Scientific, 24010043)
Trypsin-EDTA (0.05%), phenol red (Thermo Fisher Scientific, 25300054)
Transfection Reagents:
Opti-Mem (Thermo Fisher Scientific, 31985062)
Lipofectamine 3000 (Thermo Fisher Scientific, L3000008)
Dharmafect (Horizon Discovery, T-2001-01)
Falcon 5 mL Round Bottom Polystyrene Test Tube, with Cell Strainer Snap Cap (Corning, 352235)
Seeding plates
Seed 50,000 WT or GFP-mCherry-LC3B KI H4 cells in each chamber of a 6-well treated tissue culture plate. Maintain cells in DMEM + 10% fetal bovine serum (FBS) + 1x penicillin and streptomycin.
Grow for 1 day until cells are ~60-70% confluent
Transient Transfection
30m
If cells do not need to be transfected, skip to autophagy induction and cell collection
Wash cells into DMEM + 10% FBS without antibiotic prior to transfection (at 60-70% confluence).
Prepare reagents for transfection with either lipofectamine 3000 or dharmafect.
For transfected plasmids, each well requires 1 µg of target DNA construct. Prepare a master mix consisting of 1 µg of DNA, 125 µL of opti-mem low serum medium, and 2 µL of P3000 reagent per well. Prepare an additional mix consisting of 125 µL of opti-mem and 2 µL of lipofectamine 3000 per well. Combine the DNA + P3000 and lipofectamine solutions together, and allow to incubate for 00:10:00 at room temperature.
10m
For siRNA, transfect a final concentration of 25 nanomolar (nM) of the indicated siRNA. Prepare a master mix consisting of siRNA and 125 µL of opti-mem low serum medium per well. Prepare an additional mix consisting of 125 µL of opti-mem and 4 µL of Dharmafect (Horizon Discovery) per well. Combine the DNA and dharmafect solutions together, and allow to incubate for 00:10:00 at Room temperature .
10m
After 00:10:00 , dispense 250 µL of combined mixture into each well to be transfected.
10m
Return plates to incubator, and incubate for 1-3 days (minimum of 2 days for siRNA KD) before conducting experiment
Autophagy Induction and FACS analysis
4h 5m
Replace the media with Earle’s Balanced Salt Solution (EBSS) to start the experiment.
Return cells to incubator for 04:00:00 .
4h
Remove media and add 200 µL of 0.05% trypsin to each wells for 00:03:00
3m
Add 2 mL of DMEM to each well, transfer to 15 mL conical tubes, and centrifuge for 500 x g, 00:02:00 .
2m
Remove the supernatant and resuspend thoroughly in appropriate FACS buffer (we used 1x PBS supplemented with 1x FBS), pipetting up and down at least five times. Transfer to round bottom tube with cell strainer to further ensure cell singlets.
At this point, we brought the samples to a core facility for analysis. If using a core, consult with the technician prior to the experiment. The non-labeled WT sample was used as a negative control. The untreated or siControl sample was used as a positive control and should show a high ratio between the red and green fluorophores.
Data analysis was performed using the FlowJo software.
Acknowledgements
This work was supported by grants from the National Institutes of Health (R01 GM100930 and R35 GM153482 to TJM; R01 GM151829 to JB; DA018343 to PDC), F31 AG079606 to DMF and F31 DK136246 to JLK. This research was also funded in part through Aligning Science Across Parkinson’s (ASAP-025173 to TJM and PDC) through the Michael J. Fox Foundation for Parkinson’s Research (MJFF) and the Howard Hughes Medical Institute (HHMI; PDC). FS acknowledges support from the Human Frontier Science Program (LT000056/2020-C). JB acknowledges support by the Wellcome Leap Foundation. Imaging was supported by the Yale Center for Cellular and Molecular Imaging (both the fluorescence and electron microscopy facilities). We also thank the MS & Proteomics Resource at Yale University for providing the necessary mass spectrometers and the accompany biotechnology tools funded in part by the Yale School of Medicine and by the Office of The Director, National Institutes of Health (S10OD02365101A1, S10OD019967, and S10OD018034). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.