KEY FEATURES

Sensitive and accurate. Use as little as 10 μL samples. Linear detection range in 96-well plate: 0.92 to 100 μg/mL (10 to 1000 μM) glycerol for colorimetric assays and 0.2 to 5 μg/mL for fluorimetric assays.

Rapid and convenient. The procedure involves addition of a single working reagent and incubation for 20 min at room temperature.

Robust and amenable to HTS assays. Potential interference by testing drugs is greatly reduced at 570nm. Compatible with culture media containing phenol red. Assays can be performed in 96 or 384- well plates.

APPLICATIONS:

Direct Assays: adipolysis (glycerol in cell culture media).

Drug Discovery/Pharmacology: effects of testing drugs on adipolysis.

KIT CONTENTS (bulk reagents available)

Assay Buffer: 24 mL Enzyme Mix: 500 μL ATP: 250 μL

Dye Reagent: 220 μL Standard: 100 μL 100 mM Glycerol

Storage conditions. The kit is shipped on dry ice. Store Assay Buffer at 4°C and other reagents at -20°C. Shelf life of two months after receipt.

Precautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.

SH-group containing reagents (e.g. mercaptoethanol, DTT) may interfere with this assay and should be avoided in sample preparation. Prior to the assay, equilibrate all components to room temperature. Keep thawed Enzyme Mix in a refrigerator or on ice during assays.

1. Cell Culture. Note: Cells and testing drugs are to be provided by the customer and are not included in this reagent kit. Grow cells (e.g. preadipocytes, adipocytes) in culture plate (24-well, 96-well or 384- well). If desired, treat cells with testing drugs such as insulin, isoproterenol, and incubate for the desired time period.

2. Standards and Samples. Prepare a 100 μg/mL standard by mixing 10 μL 100 mM glycerol standard with 910 μL in the same medium used for cell culture. Dilute standard in the medium as follows. Transfer 10 μL standards into wells of a clear 96-well assay plate (5 μL for 384-well assay plate).

Collect cell culture supernatants from culture wells. Such samples should be assayed immediately or stored at -20°C. Transfer 10 μL samples (5 μL for 384-well assay plate) into separate wells of the assay plate.

3. Enzyme Reaction. For each assay well, mix 100 μL Assay Buffer, 2μL Enzyme Mix, 1 μL ATP and 1 μL Dye Reagent in a clean tube. Transfer 100 μL Working Reagent into each assay well. Tap plate to mix. For assays in a 384-well plate, use 50 μL Working Reagent per well.

4. Incubate 20 min at room temperature. Read optical density at 570nm (550-585nm).

Note: if the Sample OD is higher than the Standard OD at 100 μg/mL, dilute sample in water and repeat the assay. Multiply result by the dilution factor.

ODSAMPLE and ODMEDIUM are optical density values of the sample and medium (#4). Conversions: 1 μg/mL glycerol equals 10.9 μM.

FLUORIMETRIC PROCEDURE

For fluorimetric assays, the linear detection range is 0.2 to 5 μg/mL glycerol. Dilute Standards (#1 to # 4, see Colorimetric Procedure) as follows: mix 10 μL standard with 190 μL dH2O. The  glycerol concentrations are now 5.0, 3.0, 1.5 and 0 μg/mL, respectively. Cell culture supernatant: dilute by mixing 10 μL cell culture sample with 190 μL dH2O (dilution factor n = 20). Transfer 5 μL of the diluted standards and samples into separate wells of a black 96-well or 384-well plate. Add 50 μL Working Reagent and tap plate to mix. Incubate 20 min at room temperature and read fluorescence at lex = 530nm and lem = 585nm. The glycerol concentration of Sample is calculated as