TITLE: Lipid droplet visualisation in cultured cells using BODIPY 493/503 stain
AUTHORS: Laura  Smith

[DESCRIPTION]
Lipid droplets are organelles involved in intracellular lipid homeostasis, as well as playing key roles in a variety of cellular functions including cellular signalling, metabolic disease and inflammation. BODIPY™ 493/503 (4,4-Difluoro-1,3,5,7,8-Pentamethyl-4-Bora-3a,4a-Diaza-s-Indacene) is a fatty acid-conjugated fluorescent probe that has been shown to successfully visualise the localisation and dynamics of lipid droplets in cells. Analysis can be performed on cells cultured in complete growth medium or in cells following lipid loading with oleic acid (OA) after over-night starvation in OptiMEM. OA is a potent inducer of lipid droplet formation.

[STEPS]
SECTION: Basal conditions
2. Plate cells onto coverslips in 6 wells plate – want cells to be 60-80% confluent for analysis.
SECTION: Basal conditions
3. Remove media and wash 2x in PBS
SECTION: Basal conditions
4. Fix cells in 4% PFA at RT for 20 minutes.
SECTION: Basal conditions
5. Wash 1x in PBS
SECTION: Basal conditions
6. Make up BODIPY 493/503:
Stock is 1mg/ml in DMSO 
Keep away from light (always in foil)
SECTION: Basal conditions
7. Dilute BODIPY 493/503 in PBS at 1/1000 dilution
Example: if staining 21 coverslips = 250 x 21 = 5,250 uL
5.5 uL BODIPY stock in 5,500 uL PBS
SECTION: Basal conditions
8. Add 250 uL BODIPY per coverslip
SECTION: Basal conditions
9. Incubate coverslips at room temperature for 15 minutes (keep away from light)
SECTION: Basal conditions
10. Wash coverslips 3x in PBS
SECTION: Basal conditions
11. Mount coverslips with DAPI+Citifluor and analysed by fluorescence microscopy.
SECTION: Lipid loading conditions
12. Plate cells onto coverslips in 6 wells plate – want cells to be 60-80% confluent for analysis.
SECTION: Lipid loading conditions
13. Remove media from cells and replace with OptiMEM overnight
SECTION: Lipid loading conditions
14. Make up 10 mM oleic acid (1 uL OA in 170uL PBS/BSA)
SECTION: Lipid loading conditions
15. Dilute oleic 1/100 in OptiMEM for 5 hours in incubator
Example: If 1.5 mL optiMEM in well add 15 uL OA to the well
SECTION: Lipid loading conditions
16. Fix cells in 4% PFA at RT for 20 minutes.
SECTION: Lipid loading conditions
17. Wash 1x in PBS
SECTION: Lipid loading conditions
18. Make up BODIPY 493/503:
Stock is 1mg/ml in DMSO 
Keep away from light (always in foil)
SECTION: Lipid loading conditions
19. Dilute BODIPY 493/503 in PBS at 1/1000 dilution
Example: if staining 21 coverslips = 250 x 21 = 5,250 uL
5.5 uL BODIPY stock in 5,500 uL PBS
SECTION: Lipid loading conditions
20. Add 250 uL BODIPY per coverslip
SECTION: Lipid loading conditions
21. Incubate coverslips at room temperature for 15 minutes (keep away from light)
SECTION: Lipid loading conditions
22. Wash coverslips 3x in PBS
SECTION: Lipid loading conditions
23. Mount coverslips with DAPI+Citifluor and analysed by fluorescence microscopy.
SECTION: Counting lipid droplets
24. To count lipid droplets, the ImageJ software was used. The local intensity maxima of each lipid droplet was determined and counted in pre-processed images.
SECTION: Principle
1. Lipid droplets are organelles involved in intracellular lipid homeostasis, as well as playing key roles in a variety of cellular functions including cellular signalling, metabolic disease and inflammation. BODIPY™ 493/503 (4,4-Difluoro-1,3,5,7,8-Pentamethyl-4-Bora-3a,4a-Diaza-s-Indacene) is a fatty acid-conjugated fluorescent probe that has been shown to successfully visualise the localisation and dynamics of lipid droplets in cells. Analysis can be performed on cells cultured in complete growth medium or in cells following lipid loading with oleic acid (OA) after over-night starvation in OptiMEM. OA is a potent inducer of lipid droplet formation.
SECTION: Counting lipid droplets
24.1. Convert the image to a grayscale 8-bit image.

Image > Split channels (work on the image with the lipid droplets not DAPI)
Image > Type > 8-bit (repeat 2x)
SECTION: Counting lipid droplets
24.2. To ease localisation and separation of lipid droplets, subtract the background from the image. 

Process > Subtract background – change the number to the best number to not subtract any lipid droplets (my image was 25)
SECTION: Counting lipid droplets
24.3. Use the find maxima tool to count each lipid droplet in the picture.

Process > Find Maxima
Select count and click preview point selection to visualise each dot that will be measured.
Adjust noise tolerance – the higher the number the more specific so fewer dots will be selected. Want to play around with this until you get a number that is picking up most of the dots with minimal background (my image was around 15-18)
Number appears at bottom of window as x Maxima.