TITLE: An optimised protocol for the detection of lipofuscin in cells cultured in vitro
AUTHORS: Camilla SA Davan-Wetton, Trinidad Montero-Melendez

[DESCRIPTION]
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.

[GUIDELINES]
This protocol should be performed following standard laboratory practices, including the wearing of suitable PPE like laboratory coat, gloves and goggles.

[STEPS]
SECTION: Saturated Sudan Black B solution preparation
1. 
Dissolve 1.2 g Sudan Black B in 80 mL 70% ethanol in a glass bottle and stir    on a magnetic stirrer.  

SECTION: Saturated Sudan Black B solution preparation
2. 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.

 

SECTION: Nuclear Fast Red solution preparation
3. 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 .

 

SECTION: Nuclear Fast Red solution preparation
4. Dissolve 200 mg  Nuclear Fast Red in 200 mL boiling 5% aluminium sulphate solution. Boil for 5 min and allow it to return to Room temperature  before use.


SECTION: Sudan Black B staining
5. 
Remove cell culture medium and wash cells once in PBS. 


 

SECTION: Sudan Black B staining
7. Remove PFA solution and incubate cells in 70% ethanol for 2 min .


 

SECTION: Sudan Black B staining
8. iunspecificRemove ethanol and incubate cells in the freshly prepared, triple filtered Sudan Black B solution for 8 min. Place plate on a plate shaker at    for the duration of the staining. 


 

SECTION: Sudan Black B staining
9. Remove Sudan Black B solution and wash the cells in distilled water for 5 min, replacing the plate on the plate shaker at     for the duration of the washing. 

 

SECTION: A) Counterstaining with Nuclear Fast Red solution
10. Remove the distilled water and incubate cells in the Nuclear Fast Red solution for 10 min. Place the plate on the plate shaker at   for the duration of the staining.

SECTION: A) Counterstaining with Nuclear Fast Red solution
11. Remove the Nuclear Fast Red solution and wash wells in PBS for 10 min, replacing the plate on the plate shaker at   for the duration of the washing. 

SECTION: A) Counterstaining with Nuclear Fast Red solution
12. Replace the PBS with fresh PBS and visualise cells using a brightfield microscope. 
SECTION: B) Quantification of Sudan Black B staining
13. Remove the distilled water and incubate cells in 1 ug/ml DAPI solution for 10 min .
 

SECTION: B) Quantification of Sudan Black B staining
14. Remove the DAPI solution and wash the cells once in PBS for 10 min, replacing the plate on the plate shaker at   for the duration of the washing. 

SECTION: B) Quantification of Sudan Black B staining
15. 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).
SECTION: B) Quantification of Sudan Black B staining
16. 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. 


 





SECTION: Sudan Black B staining
6. Fix cells in 4% PFA for 15 min  at Room temperature.