Perilipin Screen-Certified Cell-Imaging Kit (Z’ > 0.5)
Availability: In Stock • Product size: 500 tests using 96 well plates
Perilipin is a protein that associates with lipid droplets in mature adipocytes and is involved in lipid droplet metabolism. The association of perilipin with lipid droplets in primary human adipocytes can be quantified through use of Vala Science Inc's Perilipin kit.
Item number: 4806
Kit Usage & Contents
Perilipin Kit - Instructions
Reagent A: Fixative (60 ml).
CAUTION: CONTAINS 4% PARAFORMALDEHYDE - OPEN AND USE IN A WELL VELTILATED SPACE. AVOID CONTACT AND DISPOSE OF PROPERLY
Reagent B: Permeabilizer (60 ml).
Reagent C: Blocking Buffer (100 ml)
Reagent D: Perilipin Primary Antibody (80µl)
Reagent E: Secondary Antibody (60 µl)
Reagent F: Lipid Stain. (60µl)
Reagent G: Nuclear stain (60 ml) (CAUTION: CONTAINS DAPI 4',6-Diamidino-2-phenylindole - AVOID CONTACT AND DISPOSE OF PROPERLY)
- Store kit components at 4°C
- For storage >30 days, freeze Reagent A in aliquots appropriate for your experiments.
- Component B contain 0.02% sodium azide (NaN3). Dispose of properly! Flush plumbing with excess water to avoid gas build-up.
These reagents have been validated by Vala Sciences Inc on human adipocytes, adipocytes derived from murine 3T3-L1 cells, and HeLa cells cultured in media containing fatty acids.
Reagents that are required but are not supplied with the kit include:
-96 well plate suitable for imaging (optical quality coverslip glass bottom)
-Protein substrate for coating glass bottom 96 well plates
-Phosphate buffered saline (PBS)
Protocol: The key steps are preparing the cells for the screen, performing the screen, staining the cells, imaging the cells, analyzing the data.
Figure 1. Lipid droplets in human adipocytes. Preadipocytes were cultured with 1 μM rosiglitazone, a PPARγ agonist for 14 days to promote adipogenesis. The cells were stained for lipid (green) and for nuclei (blue).
Figure 2. Lipid droplets in adipocytes derived from murine 3T3 L1 cells.
1. Preparing the cells: Use 96-well dishes featuring optical quality coverslip glass (e.g., Nunc #164588) for best results. Prior to plating, pre-coat dishes with a protein substrate (e.g., 1% gelatin that has been crosslinked with glutaraldehyde and extensively rinsed - protocol available upon request).
2. Performing the screen: Remove culture medium and replace with culture medium containing test compounds. Altering the number and size of the lipid droplets is likely to require exposure times of 1 to 7 days. Positive controls that induce extensive lipid droplet formation in adipocytes include thiazolidinediones (such as rosiglitazone).
3. Staining the cells for Perilipin and Lipid Droplets (96 well plate protocol):
- Fix cells: Remove media/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 gentle shaking. Remove Reagent B and rinse wells 2X with 200µl PBS.
-Blocking for antibody staining: Add 100µl/well Reagent C incubate at 37°C for 60 min with gentle shaking. Remove Reagent C.
- Perilipin antibody: Prepare Perilipin antibody (Reagent D) by diluting 1:200 in Reagent C (for 96 well plate 15µl Reagent D to 3ml Reagent C). Add 30ul /well and incubate at 37°C for 60 min. with gentle shaking. Only prepare enough Reagent D for the current experiment, Reagent D should be stored in undiluted form. Following incubation remove Reagent D and wash 3X with PBS. Kit provides enough Reagent D for five 96 well plates when using 30ul/well.
-Secondary Antibody: Prepare Secondary Antibody (Reagent E) by diluting 1:250 in Reagent C (for 96 well plate, 12ul Reagent E to 3 mls Reagent C). Add 30ul/well and incubate at 37°C for 60 min. with gentle shaking. Only prepare enough Reagent E for the current experiment, Reagent E should be stored in undiluted form. Following incubation remove Reagent E and wash 3X with PBS. Kit provides enough Reagent D for five 96 well plates when using 30ul/well.
-Lipid stain: Prepare Reagent F by diluting 1:333 in PBS (for 96 well plate, 9 ul in 3mls PBS). Add 30ul/well and incubate at 37°C for 30 min. Rinse 1X with PBS.
- Stain nuclei: Add 100 μl/well of Reagent G. Incubate 20 minutes at room temperature. The cells are now ready for imaging.
Figure 3. Human adipocytes labeled for lipid droplets, nuclei, and perilipin. Left panel, lipid droplets
(green) and nuclei (blue) are visualized. Right panel, perilipin (red) is visualized for the same field of view.
4. Imaging the cells: Refer to the manual of your microscopy work station 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 three fluorescence channels (blue (for DAPI), green (e.g., for FITC), and red (e.g., Texas Red)). 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). Visualize perilipin on the "red" channel (excitation at 572 nm, emitting at
5. Quantifying lipid droplet number and size can be done with the use of Vala's CyteSeerTM Lipid Droplet Analysis Software: See instruction manual and tutorials in Vala Science's CyteSeer software manual for further information. More information regarding CyteSeer software is available at http://www.valasciences.com
In addition to ordering online you can also call us on 1-888-742-8252 (VALA) or fax an order form to 1-888-742-1230. If you have any questions about this item you can also contact us through the website. Items that are in stock will be delivered in 1-2 days.
Process your cell images with CyteSeer, our automated high-content analysis software.