Screen Quest™ Fluorimetric Fatty Acid Uptake Assay Kit

Protocol summary

1. Plate cells in growth medium for 4-6 hours
2. Transfer the cells into serum free medium for 1 hour and treat cells as desired
3. Add 100 µL/well of the Fatty Acid dye-loading solution
4. Monitor fluoroscence increase at Ex/Em = 485/515 nm immediately for kinetics or after 60 minutes incubation for endpoint reading (bottom read mode)

Important notes
Thaw all the kit components at room temperature before starting the experiment.

TF2-C12 Fatty Acid stock solution:
Add 20 µL of DMSO (Component C) to the vial of TF2-C12 Fatty Acid (Component A) and mix them well. Note:  20 µL of the fluorescent fatty acid substrate stock solution is enough for one plate. The unused fluorescent fatty acid substrate stock solution can be aliquoted and stored at < -20 ^oC for up to two months if the tubes are sealed tightly and kept from light. Avoid repeated freeze-thaw cycles.

Fatty Acid dye-loading solution:
Add 20 µL of the TF2-C12 Fatty Acid stock solution to 10 mL of Assay Buffer (Component B) and mix them well. Note: 10 mL of Fatty Acid dye-loading solution is enough for one plate; prepare fresh for each plate and experiment.

Cells preparation:

Prepare cells as desired. The following protocols are guidelines to prepare 3T3-L1adipocytes.

• Prepare differentiated 3T3-L1 adipocytes (see Ref 1): 3T3-L1 fibroblasts were grown 2 days in a 75 cm flask post-confluence in DMEM/FBS, and then for 2 days in DMEM/FBS supplemented with 0.83 µM insulin, 0.25 µM dexamethasone, and 0.25 mM isobutylmethylxanthine. The medium is changed to maintain the insulin concentration with dexamethasone and IBMX absent for another 2 days. The medium was then changed to DMEM/FBS alone for another 3-5 days. Differentiated cells (at least 95% of which showed an adipocyte phenotype by accumulation of lipid droplets) were used on day 8 to 12 after induction of differentiation.
  
  
• Plate 3T3-L1 adipocytes in growth medium at 50,000-80,000 cells/well/100 µL/96-well or 12,500-20,000 cells/well/25 µL/384-well black wall/clear bottom cell culture Poly-D lysine plate for 4-6 hours before experiment. Centrifuge the plate at 800 rpm for 2 minutes with brake off. Note: It is recommended to plate 3 wells with growth medium only (without cells) as blank wells for data normalization. Note: We find that adipocytes plated at the same day (4-6 hours, and then serum deprived for 1 hour) give better results than that plated for overnight.

• Remove the cell plate from the incubator, aspirate the medium from the wells, and deprive the cells with 90 µL/well/ 96 well-plate or 20 µL/well/384 well-plate serum free medium. Incubate the cells at 37 ºC, 5% CO₂ incubator for 1 hr.
  
  
• Treat the cells by adding 10 µL/well/96-well plate (5 µL/well/384-well plate) of the test compounds or 1X Hanks and 20 mM Hepes buffer (1X HBSS, pH 7.4) or buffer of your choice as the compound diluent.  For blank wells, add the compound diluents. Incubate the cells at 37 ºC, 5% CO₂ incubator for a desired period of time (30 minutes for 3T3-L1 cells treated with Insulin).

Sample protocol:

1. Treat cells with test compounds as desired.
   
   
2. Remove compound-treated cell plates from the incubator, add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) (including blank wells) of the Fatty Acid dye-loading solution.
   
   
3. Measure the fluorescence signal with a fluorescence microplate reader at Ex/Em = 485/515 nm (cut off at 495 nm) using a bottom read mode. Note: For kinetic reading:  Read the fluorescence intensity immediately at 20 seconds interval for 30-60 minutes. Note: For endpoint reading: Read the fluorescence intensity at the end of the 30-60 minutes incubation.