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Cholesterol Quantitation Kit,IC-6609
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Unit size: 200 Tests
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Component
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Storage
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Amount
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Component A: InCellGen™ Red (light sensitive)
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Freeze (< -15 °C), Minimize light exposure
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1 vial
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Component B: Assay Buffer
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Freeze (< -15 °C)
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1 bottle (20 mL)
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Component C: Cholesterol Enzyme Mix (lyophilized)
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Freeze (< -15 °C), Minimize light exposure
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2 bottles
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Component D: Cholesterol Standard
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Freeze (< -15 °C), Minimize light exposure
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1vial (2 mM, 100 μL)
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Component E: DMSO
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Freeze (< -15 °C)
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1 vial (200 μL)
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OVERVIEW
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reaction should be performed at pH 7–8. The provided assay buffer (pH 7.4) is
recommended.
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Cholesterol is required to build and maintain cell membranes. It modulates
membrane fluidity over the range of physiological temperatures. Within cells,
cholesterol is the precursor molecule in several biochemical pathways.
Cholesterol is also an important precursor molecule for the synthesis of Vitamin
D and the steroid hormones, including the adrenal gland hormones cortisol and
aldosterone as well as the sex hormones progesterone, estrogens, together with
testosterone and their derivatives. This InCellGen™ Cholesterol Quantitation Assay
Kit provides one of the most sensitive methods for quantifying cholesterol. The kit
uses InCellGen™ Red to quantify the concentration of cholesterol, which is related
to the production of hydrogen peroxide in the cholesterol oxidase-mediated
enzyme coupling reactions in the presence of cholesterol. The amount of
cholesterol is proportional to the concentration of hydrogen peroxide formed in
the enzyme coupling reaction cycle. In the presence of peroxidase, the
fluorescence intensity of InCellGen™ Red is proportional to the concentration of
hydrogen peroxide that is converted to the concentration of cholesterol. The
assay can be readily read with a fluorescence microplate reader.
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2. Cholesterol standard stock solution (20 μM)
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Add 10 μL of Cholesterol Standard (Component D) into 990 μL of Assay Buffer
(Component B) and mix well.
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PREPARATION OF STANDARD SOLUTION
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Cholesterol standard
Prepare a cholesterol standard (20 μM). Then perform 1:3 serial dilutions in
Assay Buffer (Component B) to get approximately 10, 3, 1, 0.3, 0.1, 0.03 and
0.01 μM serially diluted cholesterol standards. A non-cholesterol buffer control is
included as blank control.
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PREPARATION OF WORKING SOLUTION
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AT A GLANCE
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Cholesterol Assay working solution
Add 5 mL of Assay Buffer (Component B) into the bottle of Cholesterol Enzyme
Mix (Component C), and mix them well. Add 20 μL of InCellGen Red ™ stock
solution (250X) into the Cholesterol Enzyme Mix bottle.
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Protocol Summary
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1. Prepare Cholesterol Assay working solution (50 μL)
2. Add cholesterol standards and/or test samples (50 μL)
3. Incubate at 37°C for 30 minutes
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SAMPLE EXPERIMENTAL PROTOCOL
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4. Monitor fluroscence intensity at Ex/Em = 540/590 nm
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Important Thaw all the kit components at room temperature before starting
the experiment.
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Table 1. Layout of Cholesterol standards and test samples in a solid black
96-well microplate. CS = Cholesterol standard (CS1-CS7); BL = blank control; TS
= test sample.
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KEY PARAMETERS
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BL
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BL
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TS
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TS
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CS1
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CS1
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...
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...
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Fluorescence microplate reader
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CS2
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CS2
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...
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...
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CS3
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CS3
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Excitation
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540 nm
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CS4
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CS4
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CS5
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CS5
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Emission
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590 nm
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CS6
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CS6
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Cutoff
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570 nm
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CS7
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CS7
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Recommended plate
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Solid black
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PREPARATION OF STOCK SOLUTIONS
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Table 2. Reagent composition for each well
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well
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Volume
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Reagent
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Unless otherwise noted, all unused stock solutions should be divided into
single-use aliquots and stored at -20 ° C after preparation. Avoid repeated
freeze-thaw cycles.
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CS1 - CS7
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50 μL
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Serial Dilutions (0.01 to 10 μM)
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BL
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50 μL
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Assay Buffer (Component B)
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TS
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50 μL
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test sample
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1. InCellGen™ Red stock solution (250X)
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Cholesterol assay
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(Component A). The stock solution should be used promptly. Any remaining
solution should be aliquoted and refrozen at -20 o C.
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1. Add cholesterol standards and cholsterol containing test samples into a 96-well solid black microplate as described in Tables 1 and 2.
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Note Avoid repeated freeze-thaw cycles.
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2. Add 50 μL of Cholesterol Assay working solution into each well of cholesterol standard, blank control, and test samples (Table 2) to make the total cholesterol assay volume of 100 μL/well. Note: For a 384-well plate, add 25 μL of sample and 25 μL of assay reactionmixture into each well.
3. Incubate the reaction for 30 minutes at 37 o C, protected from light.
4. Monitor the fluorescence intensity with a fluorescence plate reader at Ex/Em= 530-570 nm/590-600 nm (optimal Ex/Em = 540/590 nm).
Note: The contents of the plate can also be transferred to a white
clear bottom plate and read by an absorbance microplate reader at
the wavelength of 576±5 nm. The absorption detection has lower
sensitivity compared to the fluorescence reading.
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Note The InCellGen™ Red substrate is unstable in the presence of thiols such
as dithiothreitol (DTT) and 2-mercaptoethanol. The final concentration of DTT or
2-mercaptoethanol in the reaction should be no higher than 10 μM. The
InCellGen™ Red substrate is also unstable at high pH (> 8.5). Therefore, the
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