pHSLserine660 (Vala#4820)
$695.00
Availability: In Stock • Product size: 500 tests using 96 well plates
Overview
The reagents and protocol in this kit will specifically visualize Hormone Sensitive Lipase (HSL), which has been phosphorylated on serine 660 (referenced to the sequence of rat HSL). Phosphorylation of HSL on serine 660 occurs as HSL is activated in adipocytes exposed to agents that stimulate lipolysis of triglycerides. Reagents in this kit have been validated with both human and murine adipocytes, and will visualize pHSL-serine 660 in the red fluorescence channel. Lipid droplets will also be labeled, and will be visible in the green fluorescence channel.
Item number: 4820
Kit Usage & Contents
Kit Components:
Reagent A (PF02): Fixative (60 ml).
(CAUTION: CONTAINS 4% PARAFORMALDEHYDE - OPEN AND USE IN A WELL VELTILATED SPACE. AVOID CONTACT AND DISPOSE OF PROPERLY)
Reagent B (PB01): (60 ml)
Reagent C (BB01): (75 ml).
Reagent D (S703): (18 ml Reagent C; 90 ml of 200X stock) – Only prepare enough reagent D for the experiment, as it is best to store it in its undiluted form at 4 C. Prepare dilution of Reagent D by adding 5 ml of 200X stock solution per ml of Reagent C, just prior to use. Enough reagent is supplied to use 30 ml per well for five 96-well dishes.
Reagent E (F202): (18 ml Reagent C; 72 ml of 250X stock) - Only prepare enough reagent E for the experiment, as it is best to store it in its undiluted form at 4 C. Prepare dilution of Reagent E by adding 4 ml of 250X stock solution per ml of Reagent C, just prior to use. Enough reagent is supplied to use 30 ml per well for five 96-well dishes.
Reagent F (DA01): Nuclear + lipid droplets stain (60 ml) - Add 1 ml of Vala’s Lipid Stain (333X stock) per ml of Reagent F (CAUTION: CONTAINS DAPI – AVOID CONTACT AND DISPOSE OF PROPERLY)
Reagents that are required but are not supplied with the kit include:
Cells (e.g., 3T3L1 cells)
Phosphate buffered saline (PBS): (e.g., MediaTech Cat. No. 21-040-CM, PBS, 1X).
Protocol: The key steps are preparing the cells for the experiment, performing the experiment, processing the samples, imaging the cells, analyzing the data.
1. Preparing the cells for the experiment: 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., Matrigel, BD Biosciences, diluted 1/50 using “thin-coat method” as specified by manufacturer). For 3T3L1 cells, it is convenient to grow the cells to confluency in tissue flasks, add differentiation medium for 3 to 4 days to initiate the adipogenesis process, then transfer the cells to 96-well dishes at a high degree of confluency (e.g., 60,000 cells/well). Cells cultured in this manner will immediately begin expressing lipid droplets and HSL, and can be successfully labeled for HSL within 3 days of transfer to the dish; alternatively the cells can be maintained for up to 3 weeks in culture to attain full maturation to the adipocytes phenotype.
2. Prepare the reagents: Cool PBS (at least 21 ml per plate) to 4 C. Prepare test compounds (these can be prepared as either concentrated stock solutions, e.g., 10X, or at the desired final concentration (1X, which will require a full replacement of the well contents with the solution containing the test compound). Prepare a station for fixing the cells in a well ventilated place (e.g., take the bottle of Reagent A to the fume hood, setup reagent reservoirs for PBS and for Reagent A).
3. Perform the experiment:
A) Add test compounds: If drug solutions are at 1X, dump or aspirate each plate and replace well contents with test conditions. If concentrated drug stock solutions are being used, add these to the cells at this time.
B) Incubate cells for the desired period of time: Final preparations for “stopping” the cells can be made at this time (e.g., pipetting PBS and Reagent A into separate reagent reservoirs).
C) “Stop” the cells: Remove test solutions, rinse wells with 200 ml/well of cold PBS, add 100 ml of Reagent A per well. Incubate the cells with Reagent A (100 ml/well) for 15 minutes at room temperature to “fix” the cells. Rinse wells 2X with 200 ml of PBS to remove fixative.
4. Stain the cells:
- Add 100 ml/well Reagent B. Incubate at 37 C for 15 min. with rotation. Remove Reagent B.
- Add 100 ml/well Reagent C. Incubate at 37 C for 60 min. with rotation. Remove Reagent C (remove as much as possible from each well, using a multi-channel pipet or aspirator, being careful not to disturb the cell monolayer - this is particularly important for mature 3T3L1 adipocytes, which tend to be relatively non-adherent).
- Add 30 ml/well Reagent D. Incubate at 37 C for 60 min. with rotation. Remove Reagent D.
- Incubate wells with 200 ml/well PBS at 37 C for 5 min. with rotation. Remove PBS. Repeat for a total of 3 rinses.
- Add 30 ml/well of Reagent E. Incubate at 37 C for 60 min. with rotation. Remove Reagent E.
- Incubate wells with 200 ml/well PBS at 37 C for 5 min. with rotation. Remove PBS. Repeat for a total of 3 rinses.
- Add 100 ml/well of Reagent F. Incubate 20 minutes at room temperature. The cells are now ready for imaging.
5. Imaging the cells: Refer to the manual of your microscopy work station for image acquisition. Avoid overexposing the images. We typically image 3T3L1 cells using either a 20X or 40 X objective. Nuclei will be visualized on the “blue” channel (excitation filter centered at 360 nm, emitting at 460 nm). Lipid droplets will be visualized in the “green fluorescence” channel (excitation filter centered at 490 nm, emitting at 525 nm). pHSL-serine 660 will be visualized in the “red fluorescence” channel (excitation filter
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.
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