TITLE: U Mass - Leptin
AUTHORS: Jason Kim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span><span style = "font-weight:bold;"> </span></div><div class = "text-block">
This experiment provides the quantification of multiple hormones using multiplexed-Luminex technology based on beads containing specific antibodies. Sample matrices include plasma, serum, tissue culture supernatants, and tissue or cell lysates. Leptin is an adipocyte-derived hormone (adipokine) that regulates feeding behavior (hunger/satiety). Serum leptin levels are altered in obesity, insulin resistance, and type 2 diabetes.</div></div>

[STEPS]
?. Add 200 μL of Assay Buffer into each well of the plate. Seal and mix on a plate shaker for 10 minutes at room temperature (20-25°C).
?. Decant Assay Buffer and remove the residual amount from all wells by inverting the plate and tapping it smartly onto absorbent towels several times.
?. Add 10 μL of appropriate matrix solution to the background, standards, and control wells.
?. When assaying serum or plasma samples, use the Serum Matrix provided with the kit. When assaying tissue culture or other supernatant, use proper control culture medium as the matrix solution.
?. Add 10 μL of Assay Buffer to the background (0 pg/ml standard) and sample wells.
?. Add 10 μL of each Standard or Control into the appropriate wells.
?. Add 10 μL of sample into the appropriate wells.
?. Vortex Mixing Bottle and add 25 μL of the mixed or premixed beads to each well.During addition of Beads, shake bead bottle intermittently to avoid settling.
?. Seal the plate with a plate sealer. Wrap the plate with foil and incubate with agitation on a plate shaker overnight (18~20 hours) at 4°C.
?. Allow reagents and assay plate to come to room temperature. Gently remove well contents and wash plate 3x following the instructions listed in the Plate Washing section.
?. Add 50 μL of Detection Antibodies into each well. Allow the Detection Antibodies to warm to room temperature prior to addition.
?. Seal, cover with foil, and incubate with agitation on a plate shaker for 30 minutes at room temperature (20~25°C). Do not aspirate after incubation.
?. Add 50 μL Streptavidin-Phycoerythrin to each well containing the 50 μL of Detection Antibodies.
?. Seal, cover with foil, and incubate with agitation on a plate shaker for 30 minutes at room temperature (20~25°C).
?. Gently remove well contents and wash plate 3x following instructions listed in the Plate Washing section.
?. Add 150 μL of Sheath Fluid to all wells, and resuspend the beads on a plate shaker for 5 minutes.
?. Run plate on Bioplex 200, and set parameters for 125 µl and 75 beads per bead set.
?. Save and analyze the Median Fluorescent Intensity (MFI) data using a 5-parameter logistic or spline curve-fitting method for calculating cytokine/chemokines concentrations in samples. For diluted samples, multiply the calculated concentration by the dilution factor.
?. Plate Washing:
a)  Let plate “soak” on magnet for 60 seconds to allow complete settling of the magnetic beads.
b)  Remove well contents by aspiration.
c)  Wash plate with 200 μL/well of Wash Buffer
d)  Allow beads to soak for 60 seconds, and remove Wash Buffer by aspiration after each wash.
e)  Repeat wash steps as recommended in the Assay Procedure.
f)  If using the recommended plate washer for magnetic beads (Bio-Tek ELx405), follow the instructed equipment settings.