Perilipin Screen-Certified Cell-Imaging Kit (Z’ > 0.5)
Overview
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.
Kit Usage & Contents
- Store kit components at 4°C
- For storage >30 days, freeze Reagent A in aliquots appropriate for your experiments.
- Components B - E contain 0.02% sodium azide (NaN3). Dispose of properly! Flush plumbing with excess water to avoid gas build-up.
Technical tip! Adipocytes are sometimes poorly adherent to culture dishes. Rinse gently to avoid losing cells.
Kit Components:
- 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: Primary stain (20 ml base solution; 60 μl of 300X Perilipin Primary Label). Only prepare enough reagent B for the experiment, as it is best to store the Perilipin Primary Label in its's undiluted form at + 4 degrees Celsius. Prepare Reagent C by adding 3.3 μl of 300X Perilipin Primary Label per ml of base solution, just prior to use. Enough reagent is supplied to use 30 μl per well for five 96-well dishes.
- Reagent D: Secondary stain (18 ml base solution, 60 μl of 333X stock Lipid Stain, 72 μl of 250X stock Perilipin Secondary Label) - Only prepare enough reagent D for the experiment, as it is best to store the stock stolutions in their undiluted form at -4 C. Thoroughly vortex 333X Lipid Stain. Prepare Reagent D by adding 3 μl of 333X Lipid Stain plus 4 μl 250X Perilipin Secondary Stain per ml of base solution, just prior to use. Enough reagent is supplied to use 30 μl per well for five 96-well dishes.
- Reagent E: Nuclear stain (60 ml) (CAUTION: CONTAINS DAPI - AVOID CONTACT AND DISPOSE OF PROPERLY)
- Software: The latest version of CyteSeer, Vala Sciences' Automated Cell Analysis Software, can be downloaded from http://www.valasciences.com. A time limited license, unlocking CyteSeer's full capabilities, is automatically generated for any new workstation. CyteSeer is free for ongoing use as a viewer of both microscope images and CyteSeer generated cell-by-cell image mask & numeric data. Contact Vala directly for licensing details on continued automated cell analysis capabilities.
- Algorithm: Perilipin
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:
- Phosphate buffered saline (PBS): (e.g., MediaTech Cat. No. 21-040-CM, PBS, 1X).
Protocol: The key steps are preparing the cells for the screen, performing the screen, staining the cells, imaging the cells, analyzing the data.
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). Plate cells and allow to grow to near confluency. If appropriate, add media supplements to induce lipid droplet formation (for 3T3-L1 cells, culture cells several days post-confluency in DMEM +10% FBS, then supplement with 0.5 mM IBMX, 0.25 μM dexamethasone, and 1 μg/ml insulin for 3 to 4 days, then switch back to the base medium; lipid droplets will form over the next 5 days).
2. Performing the screen: Remove base 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. Stain the cells. For automated analysis of lipid droplets and perilipin it is necessary to stain the cells for lipid droplets, perilipin, and nuclei.
- 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. Remove Reagent A (dispose of properly as it contains 3.7% formaldehyde). Rinse wells 1X with 200 μl of PBS.
- Permeabilize cells: Add 100 μl/well Reagent B. Incubate at 37 C for 15 minutes with rotation. Remove Reagent B.
- Label the perilipin: Add 100 μl/well Reagent C. Incubate at 37 C for 60 minutes with rotation. Remove Reagent C. Rinse 3X with PBS. Remove PBS.
- Stain lipid droplets and perilipin: Add 100 μl/well of Reagent D. Incubate at 37 C for 60 minutes with rotation. Remove Reagent D. Rinse 3X with PBS. Rinse wells 3X with PBS. Remove PBS
- Stain the nuclei: Add 100 μl/well Reagent E. Incubate 20 minutes at room temperature. The cells are now ready for imaging.
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 630 nm).
5. Quantifying lipid droplets and perilipin: See instruction manual and tutorials in Vala Sciences CyteSeer software manual.
Ordering
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.
CyteSeer
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