Preparation of BSA complexed free fatty acids for in vitro studies
Caroline CT Tremblay, Julien Ghislain, Vincent Poitout
Abstract
This protocol describes the steps to prepare a fatty acid:BSA complex in cell culture media. It is suitable for the treatment of primary tissue (eg. isolated islets) and cell lines in ex vivo culture. Briefly, fatty acid stock solutions are prepared and complexed with BSA prior to dilution in culture media. Final fatty acid concentrations range from 0.125 to 0.5 mM. We recommend replacing the fatty acid mixture once each day throughout the treatment.
Steps
Preparation of BSA:FA complex
Preparation of Palmitate (PA) and Oleate (OA) stock solutions
As PA and OA are in powder form and very hydrostatic, handle with care as the powder will stick to everything and move about readily.
Carefully weigh out a sufficient amount of each to make 150millimolar (mM) stock solutions in ethanol.
MW (PA) = 278.41, stored in a desiccator at 4°C.
MW (OA) = 305, stored in a desiccator at -20°C.
Preparation of 1mL of 150millimolar (mM) stock solution:
PA Weight out 41.8mg and put in a 1.5 ml tube.
Add 1mL of 50% (v/v) ethanol.
Vortex and heat at65°C until dissolved; vortex periodically.
OA Weight out 45.7mg and put in a 1.5 ml tube.
Add 500µL of 100% (v/v) ethanol.
Vortex and heat at 65°C for 0h 15m 0s; mix occasionally.
Then add 500µL of MilliQ water, and vortex. The remaining powder should dissolve instantly.
Return to 65°C until ready for use.
Preparation of 0.5 mM fatty acid solution in culture media (FA:BSA at 5:1 molar ratio)
Prepare 10% (v/v) fatty acid-free BSA:
Weight 5g and dissolve in50mL of MilliQ water.
Filter solution with a Millipore Steriflip-GP Sterile Centrifuge Tube Top Filter Unit.
Store at 4°C.
Under a laminar flow hood, add the following components per ml of culture media:
Place 67µL of 10% (v/v) fatty acid-free BSA into a 15 ml Falcon tube.
Heat in 37°C water bath for 0h 5m 0s.
Add 3.3µL of the 150millimolar (mM) fatty acid stock solution.
Return to the 37°C water bath for 0h 5m 0s. Visually check for cloudiness. If clear, great, if cloudy, start over.
Incubate in the 37°C water bath for 1h 0m 0s.
Then add 930µLof culture medium (eg. RPMI 1640 plus 10% (v/v) FBS) heated to 37°C.
For control tube (Vehicle/BSA), add 3.3µL of 50% (v/v) ethanol to 67µL of 10% (v/v) fatty acid-free BSA into a 15 ml Falcon tube, and treat as above.
Preparation of 0.15 mM fatty acid solution in culture media (FA:BSA at 1.5:1 molar ratio)
To prepare a solution with a final fatty acid concentration of 0.125millimolar (mM) proceed as described in step 2 adding 1µL of the 150millimolar (mM) fatty acid stock solution (instead of 3.3µL). Also reduce the ethanol to 1µL for the control.
To prepare any fatty acid concentration up to0.5millimolar (mM) final adjust the amount of fatty acid stock solution accordingly while keeping the BSA constant.
