Jun 18, 2024

Public workspaceAn optimised protocol for the detection of lipofuscin in cells cultured in vitro

PLOS One
Peer-reviewed method
DOI
https://dx.doi.org/10.17504/protocols.io.x54v9yw91g3e/v1
An optimised protocol for the detection of lipofuscin in cells cultured in vitro
  • Camilla SA Davan-Wetton1,
  • Trinidad Montero-Melendez1
  • 1The William Harvey Research Institute, Queen Mary University of London, London EC1M 6BQ, United Kingdom
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Montero-Melendez Group
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DOI: https://dx.doi.org/10.17504/protocols.io.x54v9yw91g3e/v1
External link: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0306275
Protocol Citation: Camilla SA Davan-Wetton, Trinidad Montero-Melendez 2024. An optimised protocol for the detection of lipofuscin in cells cultured in vitro. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9yw91g3e/v1
Manuscript citation:
Davan-Wetton CSA, Montero-Melendez T (2024) An optimised protocol for the detection of lipofuscin, a versatile and quantifiable marker of cellular senescence. PLoS ONE 19(7): e0306275. https://doi.org/10.1371/journal.pone.0306275
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 27, 2022
Last Modified: March 07, 2025
Protocol Integer ID: 63346
Keywords: Senescence, lipofuscin, lysosomes, immunofluorescence, ageing, microscopy, lipofuscin in cell, using fluorescence microscopy, based immunofluorescence, fluorescence microscopy, brightfield microscopy, marker of cellular senescence, staining technique, fluorescence, lysosome, cell, conventional antibody, inexpensive method for the staining, cellular senescence,
Funders Acknowledgements:
Barts Charity
Grant ID: G-002392
Biotechnology and Biological Sciences Research Council (BBSRC)
Grant ID: BB/M009513/1
Disclaimer
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Abstract
Lipofuscin is a complex material that accumulates in lysosomes and it is considered a marker of cellular senescence and ageing. This substance can be detected using the lipophilic stain Sudan Black B. This protocol describes an easy, straightforward and inexpensive method for the staining of lipofuscin using Sudan Black B and imaging via brightfield microscopy. Additionally, this protocol offers an alternative method for detection of Sudan Black B stained lipofuscin using fluorescence microscopy, which facilitates co-staining of Sudan Black B with conventional antibody-based immunofluorescence staining techniques.
Image Attribution
Image credit: Camilla SA Davan-Wetton
Guidelines
This protocol should be performed following standard laboratory practices, including the wearing of suitable PPE like laboratory coat, gloves and goggles.
Materials

Reagents and Solutions

Reagent4% Paraformaldehyde solution
ReagentSudan Black BMerck MilliporeSigma (Sigma-Aldrich)
Reagent70% Ethanol
ReagentAluminium sulphateMerck MilliporeSigma (Sigma-Aldrich)
ReagentPhosphate-buffered saline
ReagentNuclear Fast RedMerck MilliporeSigma (Sigma-Aldrich)
Reagent4′,6-diamidino-2-phenylindole (DAPI)Merck MilliporeSigma (Sigma-Aldrich)


Materials

  • Cell strainer pore size 70μm
  • PDVF syringe filter pore size 0.45μm
  • PDVF syringe filter pore size 0.22μm
  • 20ml syringes
  • Magnetic stirring bars

Equipment


Equipment
Magnetic Stirrer, Lab Disc
NAME
Magnetic Stirrer
TYPE
VWR
BRAND
442-0883
SKU


Equipment
FTA-1
NAME
Aspirator with trap flask
TYPE
Biosan
BRAND
BS-040108-AAK
SKU

Equipment
PMS-1000i
NAME
Microplate Shaker
TYPE
Grant Bio
BRAND
SHA7910
SKU

Equipment
EVOS XL Core Imaging System
NAME
Bright field microscope
TYPE
ThermoFisher Scientific
BRAND
AMEX1000
SKU

Equipment
EVOS® FL Auto Imaging System
NAME
Fluorescence microscope
TYPE
ThermoFisher Scientific
BRAND
AMEX1000
SKU
Equipped with Cy5 (628/40 nm Excitation; 692/40 nm Emission) and DAPI (357/44 nm Excitation; 447/60 nm Emission) Light Cubes
SPECIFICATIONS
Software

  • Image processing package Fiji (ImageJ2, version 2.14.0/1.54f)



Troubleshooting
Safety warnings
It is recommended to familiarise yourself with the safety data sheets (SDS) relevant to this method and to conduct the corresponding risk assessment before commencing the protocol. For chemical hazards, follow appropriate safety precautions and waste disposal methods as per the local guidelines and regulations in your area.
Before start
This protocol is optimised for the staining of cells cultured in vitro in 24-well plates. Plate your cells and treat with appropriate compounds or stimuli for the required time. Remember to prepare the Saturated Sudan black B solution (steps 1-2) the day before you plan to stain your cells, as this solution requires to be stirred overnight for complete dissolution.
Saturated Sudan Black B solution preparation
16h

Dissolve Amount1.2 g Sudan Black B in Amount80 mL 70% ethanol in a glass bottle and stir DurationOvernight on a magnetic stirrer.

Before use, filter the solution three times as follows:
- first through a 70μm cell strainer
- then through a 0.45μm syringe filter
- finally through a 0.22μm syringe filter.

Note
Prepare Sudan Black solution fresh. Storing and re-using old solutions is not recommended.

Nuclear Fast Red solution preparation
Prepare a 5% aluminium sulphate solution by dissolving Amount25 g aluminium sulphate in Amount500 mL of distilled water. Stir until dissolved, sterilise by filtration using a 0.22μm filter and store at TemperatureRoom temperature .

Note
Sterilisation is recommended when preparing a larger volume of aluminium sulphate to prevent microorganism growth, as a 500ml batch can last for several months.

Dissolve Amount200 mg Nuclear Fast Red in Amount200 mL boiling 5% aluminium sulphate solution. Boil for Duration00:05:00 and allow it to return to TemperatureRoom temperature before use.


5m
Sudan Black B staining

Remove cell culture medium and wash cells once in PBS.


Note
For this and subsequenct steps where solutions are removed, an aspirator with a trap flask can be used.

Fix cells in 4% PFA for Duration00:15:00 at TemperatureRoom temperature .



Note
PFA is a chemical hazard and needs to be used inside a chemical fume hood.




15m
Toxic
Remove PFA solution and incubate cells in 70% ethanol for Duration00:02:00 .


Note
PFA is a chemical hazard and needs to be disposed of in an appropriate waste container.

2m
Toxic
iunspecificRemove ethanol and incubate cells in the freshly prepared, triple filtered Sudan Black B solution for Duration00:08:00 . Place plate on a plate shaker at Shaker200 rpm for the duration of the staining.


Note
Shaking the plate during the staining process is strongly advised to avoid SBB precipitates which will produce unspecific staining.

8m
Remove Sudan Black B solution and wash the cells in distilled water for Duration00:05:00 , replacing the plate on the plate shaker at Shaker200 rpm for the duration of the washing.

Note
Sudan Black B solution should be disposed of according to local waste management regulations and should not enter the drainage system.

At this stage, the protocol is subdivided depending if qualitative (A) or quantitative (B) staining is desired.

5m
A) Counterstaining with Nuclear Fast Red solution
Remove the distilled water and incubate cells in the Nuclear Fast Red solution for Duration00:10:00 . Place the plate on the plate shaker at Shaker200 rpm for the duration of the staining.

10m
Remove the Nuclear Fast Red solution and wash wells in PBS for Duration00:10:00 , replacing the plate on the plate shaker at Shaker200 rpm for the duration of the washing.

10m
Replace the PBS with fresh PBS and visualise cells using a brightfield microscope.
B) Quantification of Sudan Black B staining
20m
Remove the distilled water and incubate cells in Amount1 ug/ml DAPI solution for Duration00:10:00 .
Note
The plate should be protected from light by covering with aluminium foil from now onwards as DAPI is light sensitive.


10m
Remove the DAPI solution and wash the cells once in PBS for Duration00:10:00 , replacing the plate on the plate shaker at Shaker200 rpm for the duration of the washing.

10m
Replace the PBS with fresh PBS and visualise cells using a fluorescence microscope. Sudan Black B can be visualised with a Cy5 filter (far-red, 628/40 nm Excitation; 692/40 nm Emission).
Quantification may be performed, either by calculating the proportion of fluorescent cells (i.e. percentage of positive cells), or by measuring the total fluorescence intensity per cell.


Note
Total fluorescence can be quantified in Fiji using the 'Integrated Density' measurement.