Lipid droplets are the primary fat storage organelles of fat cells (adipocytes). Lipid droplet size, and number are altered by anti-diabetic and anti-obesity drugs and these effects can be quantified utilizing Vala Science Inc’s Lipid Droplet Kit. The kit can be utilized to quantify lipid droplets in human primary adipocytes, murine 3T3L1 adipocytes, as well as primary or immortalized hepatocytes.
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Sufficient for five 96-well dishes.
Reagent A: Fixative 60 ml
CAUTION: CONTAINS 4% PARAFORMALDEHYDE – OPEN AND USE IN A WELL-VENTILATED SPACE. AVOID CONTACT AND DISPOSE OF PROPERLY
Reagent B: Permeabilizer 60 ml
Reagent C: Lipid stain 18 ml base solution; 60 μl of 333X stock
Reagent D: Nuclear stain 60 ml
- Fix cells: Remove test solutions, rinse wells with 200 μl/well of cold PBS, add 100 μl of Reagent A per well. Incubate the cells with Reagent A for 15 minutes at room temperature. Rinse wells 1X with 200 μl of PBS to remove fixative.
- Permeabilize cells: Add 100 μl/well Reagent B. Incubate at 37 C for 15 mins with rotation. Remove Reagent B.
- Stain the lipid droplets: Vortex Reagent C 333X stock solution thoroughly. Prepare dilution of Reagent C by adding 3 μl of 333X stock solution per ml of base solution, just prior to use. Add 100 μl/well Reagent C. Incubate at 37 C for 60 mins with rotation. Remove Reagent C. Rinse 3X with PBS.
- Stain nuclei: Add 100 μl/well of Reagent D. Incubate 20 minutes at room temperature. The cells are now ready for imaging.
Refer to the manual of your microscopy workstation for image acquisition. Avoid overexposing the images. We typically image adipocytes using a 40X objective. Sharp focus is critical! For each field of view, make sure to image the cells in two fluorescence channels (blue and green). Visualize nuclei on the “blue” channel (excitation filter centered at 360 nm, emitting at 460 nm). Visualize lipid droplets on the “green” channel (excitation at 490 nm, emitting at 520 nm).