May 02, 2022

Public workspacePreparation of BSA complexed free fatty acids for in vitro studies

DOI
https://dx.doi.org/10.17504/protocols.io.b6r2rd8e
Preparation of BSA complexed free fatty acids for in vitro studies
  • Caroline Tremblay1,
  • Julien Ghislain1,
  • Vincent Poitout2
  • 1Montreal Diabetes Research Center, CRCHUM, Montréal, QC, Canada.;
  • 2Montreal Diabetes Research Center, CRCHUM, and Department of Medicine, Université de Montréal, Montréal, QC, Canada.
Julien Ghislain
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DOI: https://dx.doi.org/10.17504/protocols.io.b6r2rd8e
Protocol Citation: Caroline Tremblay, Julien Ghislain, Vincent Poitout 2022. Preparation of BSA complexed free fatty acids for in vitro studies. protocols.io https://dx.doi.org/10.17504/protocols.io.b6r2rd8e
Manuscript citation:
Briaud I, Harmon JS, Kelpe CL, Segu VB, Poitout V. Lipotoxicity of the pancreatic beta-cell is associated with glucose-dependent esterification of fatty acids into neutral lipids. Diabetes. 2001 Feb;50(2):315-21. doi:10.2337/diabetes.50.2.315. PMID: 11272142.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: March 25, 2022
Last Modified: May 02, 2022
Protocol Integer ID: 59930
Keywords: fatty acids, BSA complex, cell culture, bsa complex in cell culture media, fatty acid stock solution, preparation of bsa, free fatty acid, fatty acid, final fatty acid concentration, ex vivo culture, cell culture media, cell lines in ex vivo culture, isolated islet, bsa complex, preparation
Funders Acknowledgements:
CIHR
Grant ID: 0406014428
NIH
Grant ID: 5R01DK058096-15
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.
Materials
ReagentMilliQ Water
ReagentEthanol, AnhydrousBio Basic Inc.Catalog #D0193
ReagentSteriflip-GP Sterile Centrifuge Tube Top Filter UnitMerck Millipore (EMD Millipore)Catalog #SCGP00525
Reagentsodium oleateMerck MilliporeSigma (Sigma-Aldrich)Catalog #O7501
ReagentSodium palmitateMerck MilliporeSigma (Sigma-Aldrich)Catalog #P9767
ReagentFatty Acid Free Heat Shock Bovine Serum Albumin Powderequitech bio, inc.Catalog #BAH66-0500

Troubleshooting
Preparation of BSA:FA complex
1h 25m
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 Concentration150 millimolar (mM) stock solutions in ethanol.
MW (PA) = 278.41, stored in a desiccator at Temperature4 °C .
MW (OA) = 305, stored in a desiccator at Temperature-20 °C .

Preparation of Amount1 mL of Concentration150 millimolar (mM) stock solution:

PA Weight out Amount41.8 mg and put in a 1.5 ml tube.
Add Amount1 mL of Concentration50 % (v/v) ethanol.
Vortex and heat atTemperature65 °C until dissolved; vortex periodically.
OA Weight out Amount45.7 mg and put in a 1.5 ml tube.
Add Amount500 µL of Concentration100 % (v/v) ethanol.
Vortex and heat at Temperature65 °C for Duration00:15:00 ; mix occasionally.
Then add Amount500 µL of MilliQ water, and vortex. The remaining powder should dissolve instantly.
Return to Temperature65 °C until ready for use.
15m
Preparation of 0.5 mM fatty acid solution in culture media (FA:BSA at 5:1 molar ratio)

Prepare Concentration10 % (v/v) fatty acid-free BSA:
Weight Amount5 g and dissolve inAmount50 mL of MilliQ water.
Filter solution with a Millipore Steriflip-GP Sterile Centrifuge Tube Top Filter Unit.
Store at Temperature4 °C .
Under a laminar flow hood, add the following components per ml of culture media:
Place Amount67 µL of Concentration10 % (v/v) fatty acid-free BSA into a 15 ml Falcon tube.
Heat in Temperature37 °C water bath for Duration00:05:00 .
Add Amount3.3 µL of the Concentration150 millimolar (mM) fatty acid stock solution.
Return to the Temperature37 °C water bath for Duration00:05:00 . Visually check for cloudiness. If clear, great, if cloudy, start over.
Incubate in the Temperature37 °C water bath for Duration01:00:00 .
Then add Amount930 µL of culture medium (eg. RPMI 1640 plus Concentration10 % (v/v) FBS) heated to Temperature37 °C .
For control tube (Vehicle/BSA), add Amount3.3 µL of Concentration50 % (v/v) ethanol to Amount67 µL of Concentration10 % (v/v) fatty acid-free BSA into a 15 ml Falcon tube, and treat as above.
1h 10m
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 Concentration0.125 millimolar (mM) proceed as described in step 2 adding Amount1 µL of the Concentration150 millimolar (mM) fatty acid stock solution (instead of Amount3.3 µL ). Also reduce the ethanol to Amount1 µL for the control.
To prepare any fatty acid concentration up toConcentration0.5 millimolar (mM) final adjust the amount of fatty acid stock solution accordingly while keeping the BSA constant.