Lipid Droplet Research

∼ Feature ∼

  • High selectivity to lipid droplet (low background)
  • 3 color available (blue, green and red)
  • High intracellular retentivity
  • Applicable to both live and fixed cell


 


Product Description

Lipid droplets (LDs) are composed of neutral lipids such as triacylglycerol & cholesteryl ester that are surrounded by phospholipid monolayers and are seen ubiquitously, not only in adipocytes1). Although LDs were simply thought to serve as a lipid storage unit, a recent study has stated that LDs play an important role in regulating lipid metabolism, autophagy2) and cellular senescence3). Lipi probes are small molecule which emit strong fluorescence in hydrophobic environment such as in LDs. LDs can be observed without any washing steps after staining with Lipi probes.

1) T. Fujimoto et al., “Lipid droplets: a classic organelle with new outfits.” Histochem Cell Biol., 2008, 130(2), 263.
2) R. Singh et al., “Autophagy regulates lipid metabolism.” Nature, 2009, 458(7242), 1131.

Oleic acid treated HeLa cells with Lipi Product Series


<Staining Condition>
A medium that contained oleic acid (200 μmol/l) was added and incubated overnight. Then, the supernatant was removed and the cells were washed with PBS. Each Lipi product series (1 μmol/l) was added and the cells were incubated for 15 minutes.

<Detection Condition>
Lipi-Blue: Ex. 405 nm / Em. 450 - 500 nm
Lipi-Green: Ex. 488 nm / Em. 500 - 550 nm
Lipi-Red: Ex. 561 nm / Em. 565 - 650 nm

Reagent Comparison

*Leaks in GFP filter


High Correlation with Antibody Detection Method

After fixing HepG2 with 4% PFA, cells are stained with 100nmol/l Lipi-Green. Then, Adipophilin (ADFP) expressed on lipid-droplet membrane was labeled with anti-ADFP antibody.


Scale Bar: 20 μm
<Detection Condition>
Lipi-Red: Ex. 561 nm / Em. 565 - 650 nm
Anti-ADFP antibody (Alexa Fluor® 647): Ex. 640 nm / Em. 650 - 700 nm

High Selectivity toward Lipid Droplet

Live HeLa cells were treated with Oleic acid and were stained with 100 nmol/l Lipi-Green and 100 nmol/l Nile Red. Nile red had high background due to the limit in selectivity toward lipid droplets.


<Detection Condition>
Lipi-Red: Ex. 561 nm / Em. 565 - 650 nm
Nile Red: Ex. 561 nm / Em. 565 - 650 nm

Filter Leakage Rate (Lipi-Red vs Nile Red)

HepG2 cells were stained with Lipi-Red and Nile Red. Lipi-Red was imaged with Green excitation (G), but not Blue excitation (B). However, Nile Red was imaged in both filter. Lipi-Red is preferable for multi-staining.



High Intracellular Retentivity

Live HepG2 cells were stained with each of the Lipi products, Nile Red, and Reagent B.

Scale Bar: 20 μm

Lipi-Blue and Lipi-Green had higher retention in cells after 24 hours post staining than Lipi-Red, Nile Red, and Reagent B.


Recommended Filter: Wavelength for Excitation and Emission



Storage Condition: ambient temperature, protect from light
Shipping Condition: ambient temperature


Questions:

Q1: What should I do if the fluorescence is not detected?
Q2: Can I use Lipi-dye for fixed cells?

Answers:

Q1: What should I do if the fluorescence is not detected?

1. Filter conditions

Check the fluorescence spectrum of the dye on the product HP and confirm if the excitation / emission wavelength for your filter is suitable.

2. Concentration of Lipi probe working solution

Optimize the concentration within the following range:
Lipi-Blue, Lipi-Green: 0.1-0.5 μmol / L
Lipi-Red: 1-5 μmol / L

* If fluorescence is not detected even under the above conditions, prepare a higher concentration of the working solution.
Lipi-Blue & Lipi-Green: 1-2 μmol / L
Lipi-Red: 10 - 20 μmol / L

3. Incubation time
Incubate for 1-2 hours after adding Lipi probe working solution.

4. Assay conditions (fixed cells)
Please refer to Q2.

5. Other
Depending on the cell type, lipid droplets can be smaller than usual and it may be too difficult to observe.

If that is the case, please observe under a high magnification microscope or prepare for positive control with oleic acid treated cells.

Q2: Can I use Lipi-dye for fixed cells?
Yes, Lipi-dye can be used for fixed cells.

* Please use paraformaldehyde (PFA) for fixation. Alcohol fixation is not recommended because it may affect the structure of lipid droplets.

* Depending on the cell, it may not be stained or weakened in sensitivity due to fixed conditions before and after staining. In that case, please consider fixed conditions.

Fix cells after staining < HepG2 cells>
1.Remove the medium and wash twice with PBS.
2.Add Lipi-dye Working solution(in PBS) in the cells and incubated at 37 ℃ for 15 minutes.
3. Remove the supernatant and wash twice with PBS.
4. Add 2 ml of 4% paraformaldehyde (PFA) /PBS solution to the cells and incubate at room temperature for 5 minutes.
5. Remove the supernatant and wash with PBS.
6. Observe under a fluorescence microscope.

Fix cells before staining <HeLa cell>
1. Remove the medium and wash twice with PBS.
2. Add 2 ml of 4% paraformaldehyde (PFA) /PBS solution to the cells and incubate at room temperature for 5 minutes.
3. Remove the supernatant and wash twice with PBS.
4. Add Lipi-dye Working solution(in PBS) in the cells and incubated at 37 ℃ for 15 minutes.
5. Remove the supernatant and wash with PBS.

6. Observe under a fluorescence microscope.

Item # Description/Size Availability Qty Break Price Quantity
LD03-10
100 nmol
1-2 business days 1 $180.00

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For Research Use Only Products