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
Biotechnology and Biological Sciences Research Council (BBSRC)
Grant ID: BB/M009513/1
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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.
Image processing package Fiji (ImageJ2, version 2.14.0/1.54f)
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
Saturated Sudan Black B solution preparation
16h
Dissolve 1.2 g Sudan Black B in 80 mL 70% ethanol in a glass bottle and stir Overnight 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.
Nuclear Fast Red solution preparation
Nuclear Fast Red solution preparation
Prepare a 5% aluminium sulphate solution by dissolving 25 g aluminium sulphate in 500 mL of distilled water. Stir until dissolved, sterilise by filtration using a 0.22μm filter and store at Room temperature .
Dissolve 200 mg Nuclear Fast Red in 200 mL boiling 5% aluminium sulphate solution. Boil for 00:05:00 and allow it to return to Room temperature before use.
5m
Sudan Black B staining
Sudan Black B staining
Remove cell culture medium and wash cells once in PBS.
Fix cells in 4% PFA for 00:15:00 at Room temperature.
15m
Remove PFA solution and incubate cells in 70% ethanol for 00:02:00 .
2m
iunspecificRemove ethanol and incubate cells in the freshly prepared, triple filtered Sudan Black B solution for 00:08:00. Place plate on a plate shaker at 200 rpm for the duration of the staining.
8m
Remove Sudan Black B solution and wash the cells in distilled water for 00:05:00, replacing the plate on the plate shaker at 200 rpm for the duration of the washing.
5m
A) Counterstaining with Nuclear Fast Red solution
A) Counterstaining with Nuclear Fast Red solution
Remove the distilled water and incubate cells in the Nuclear Fast Red solution for 00:10:00. Place the plate on the plate shaker at 200 rpm for the duration of the staining.
10m
Remove the Nuclear Fast Red solution and wash wells in PBS for 00:10:00, replacing the plate on the plate shaker at 200 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
B) Quantification of Sudan Black B staining
20m
Remove the distilled water and incubate cells in 1 ug/ml DAPI solution for 00:10:00 .
10m
Remove the DAPI solution and wash the cells once in PBS for 00:10:00, replacing the plate on the plate shaker at 200 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.