05 Cell Staining

Lipid Droplet Assay Kit - Deep Red

Lipid Droplet Assay

Product code

LD06　 Lipid Droplet Assay Kit - Deep Red

Unit size	Price	Item Code
1 set *		LD06-10

* The typical number of usable assays : 96 well plate x 1, 40 assays for flow cytometry

Component

1 set *	・Staining Dye - Deep Red ・Loading Buffer (10x)	×1 6 ml ×1

C of A

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Product Description

Lipi probes are small molecule which emit strong fluorescence in hydrophobic environment such as in LDs.

Manual

Manual

Technical info

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⁾.Therefore, have gained great attention as an important tool to elucidate the mechanisms of formation, growth, fusion, and retraction of LDs.

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.
3) M. Yokoyama et al., “Inhibition of endothelial p53 improves metabolic abnormalities related to dietary obesity.” Cell Reports, 2014, 7(5), 1691.

Only by the addition of a reagent, the imaging of lipid droplets (LDs) or the quantitative variation of LDs in live and fixed cells becomes quantifiable.

Lipid Droplet Assay Kit considerably shortens the entire process and can be used for live cells.
The fluorescent dye provided in the Lipid Droplet Assay Kit can be used for live and fixed cells. Compared to a method of using a colorimetric reagent, the method of using the Lipid Droplet Assay Kit can shorten measuring time. Furthermore, the repeatability of experiment can be increased by using the Lipid Droplet Assay Kit because the dye is not deposited in a plate.

Experimental example of plate assay

Changes in lipid droplets were examined after the addition of oleic acid or Triacsin C (acyl-CoA synthetase inhibitor) to the A549 cell culture medium.

As a result, we confirmed that the oleic acid-treated cells show an increase in the number of LDs, compared to control and Triacsin C-treated cells.

Blue　　：Ex. 376 – 386 nm / Em 435 – 455 nm
Deep Red ：Ex. 623 – 633 nm / Em 649 – 669 nm

Reagent Comparison

Changes in lipid droplets were examined after the addition of oleic acid or Triacsin C (acyl-CoA synthetase inhibitor) to the HeLa cell culture medium.
As a result, we confirmed that the oleic acid-treated cells show an increase in the number of LDs, compared to control and Triacsin C-treated cells.

Blue　　：Ex. 405 nm/ Em 425 – 475 nm
Deep Red ：Ex. 640 nm/ Em 650 – 670 nm

Related Product Information

Function	Product	Size	Product Code
Imaging	Lipi-Blue	10 nmol	LD01
	Lipi-Green	10 nmol	LD02
	Lipi-Red	100 nmol	LD03
	Lipi-Deep Red	10 nmol	LD04
Quantification (Plate Reader, FCM)	Lipid Droplet Assay Kit - Blue	1 set	LD05
Quantification (Plate Reader, FCM)	Lipid Droplet Assay Kit - Deep Red	1 set	LD06

Q & A

Q Can I use Lipi series for fixed cells?: A

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
1. Remove the medium and wash twice with PBS.
2. Add Lipi series Working solution (in PBS) in the cells and incubated at 37 ℃ for 30 minutes.
3. Remove the supernatant and wash twice with PBS.
4. Add 4% paraformaldehyde (PFA) /PBS solution to the cells and incubate at room temperature for 5 minutes.
5. Remove the supernatant and wash with PBS.
○ Fix cells before staining
1. Remove the medium and wash twice with PBS.
2. Add 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.

Q Preparing a stock solution of oleic acid: A

Required Reagents:
・BSA (bovine serum albumin)
・Oleic acid
・0.1 mol/L Tris-HCl (pH 8.0)
Procedure:
(1) Dissolve 0.14 g/mL BSA in 0.1 mol/L Tris-HCl (pH 8.0).
(2) Add 4 mmol/L oleic acid to a disposable centrifuge tube.
(3) Add BSA solution (prepared in step 1).
(4) Cap the tube and mix on a rotary shaker (Be sure the solution is transparent, indicating that oleic acid has been conjugated to BSA).
(4) Filter the solution prepared above (step 4) using 0.22μm filter membranes.
(5) Store oleic acid stock solution at 4˚C.
*Use the appropriate amount of oleic acid stock solution for culture medium to prepare working solution.
*Oleic acid working solution cannot be stored. Please prepare the working solution immediately before usage.

Inducing lipid droplets
(1) Incubate cells for 24 hours at 37˚C in a 5% CO₂ atmosphere.
(2) Add 200 µmol/L working solution (prepared from oleic acid stock solution) to culture medium and incubate for further 24 hours.

Handling and storage condition

Handling and storage condition
0-5°C

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Lipid Droplet Assay Kit - Deep Red

Lipid Droplet Assay Kit - Deep Red