An optimised protocol for the detection of lipofuscin in cells cultured in vitro
Camilla SA Davan-Wetton, Trinidad Montero-Melendez
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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.
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.
Steps
Saturated Sudan Black B solution preparation
Dissolve 1.2g Sudan Black B in 80mL 70% ethanol in a glass bottle and stir 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
Prepare a 5% aluminium sulphate solution by dissolving 25g aluminium sulphate in 500mL of distilled water. Stir until dissolved, sterilise by filtration using a 0.22μm filter and store at Room temperature .
Dissolve 200mg Nuclear Fast Red in 200mL boiling 5% aluminium sulphate solution. Boil for 0h 5m 0s and allow it to return to Room temperature before use.
Sudan Black B staining
Remove cell culture medium and wash cells once in PBS.
Fix cells in 4% PFA for 0h 15m 0s at Room temperature.
Remove PFA solution and incubate cells in 70% ethanol for 0h 2m 0s .
iunspecificRemove ethanol and incubate cells in the freshly prepared, triple filtered Sudan Black B solution for 0h 8m 0s. Place plate on a plate shaker at 200rpm for the duration of the staining.
Remove Sudan Black B solution and wash the cells in distilled water for 0h 5m 0s, replacing the plate on the plate shaker at 200rpm for the duration of the washing.
A) Counterstaining with Nuclear Fast Red solution
Remove the distilled water and incubate cells in the Nuclear Fast Red solution for 0h 10m 0s. Place the plate on the plate shaker at 200rpm for the duration of the staining.
Remove the Nuclear Fast Red solution and wash wells in PBS for 0h 10m 0s, replacing the plate on the plate shaker at 200rpm for the duration of the washing.
Replace the PBS with fresh PBS and visualise cells using a brightfield microscope.
B) Quantification of Sudan Black B staining
Remove the distilled water and incubate cells in 1ug/ml DAPI solution for 0h 10m 0s .
Remove the DAPI solution and wash the cells once in PBS for 0h 10m 0s, replacing the plate on the plate shaker at 200rpm for the duration of the washing.
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.
