Lipoprotein Fractionation in Juvenile Mice

Lipoprotein fractions in juvenile mice were assessed by applying serum (100 μl per animal) directly to three Superdex 200 gel filtration columns (10 × 300 mm; GE Healthcare Life Sciences, Chicago, IL) arranged in series on an ÄKTA FPLC system (GE Healthcare, Chicago, IL). Samples were processed at a flow rate of 0.3 mL/min in standard Tris buffer (10 mM Tris, 0.15 M sodium chloride, 1 mM EDTA, and 0.2% sodium azide). Eluate was collected as 47 1.5-mL fractions on a Frac-900 Fraction Collector (GE Healthcare) at 4°C. Apolipoprotein B-containing lipoproteins VLDL and LDL elute as a single peak between fractions 14 and 18 (peak 1); HDL-sized particles elute as a broader peak between fractions 19 and 26 (peak 2) (28 (link), 29 (link)). Fractions were assessed for TC by colorimetric kits (Wako, Cape Charles, VA).

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Atreya M.R., Whitacre B.E., Cvijanovich N.Z., Bigham M.T., Thomas N.J., Schwarz A.J., Weiss S.L., Fitzgerald J.C., Allen G.L., Lutfi R., Nowak J.E., Quasney M.W., Shah A.S, & Wong H.R. (2020). Proprotein Convertase Subtilisin/Kexin Type 9 Loss-of-Function Is Detrimental to the Juvenile Host With Septic Shock. Critical care medicine, 48(10), 1513-1520.

Publication 2020

Superdex 200 gel filtration columns ÄKTA FPLC system Frac-900 Fraction Collector

Animal Apolipoprotein b Colorimetric Edta Gel filtration Lipoproteins Mice Serum Sodium azide Sodium chloride Tris

Corresponding Organization :

Other organizations : Cincinnati Children's Hospital Medical Center, University of Cincinnati Medical Center, UCSF Benioff Children's Hospital, Akron Children's Hospital, Penn State Milton S. Hershey Medical Center, Children's Hospital of Orange County, Children's Hospital of Philadelphia, Children's Mercy Hospital, Riley Hospital for Children, Children's Minnesota, C. S. Mott Children's Hospital, University of Michigan–Ann Arbor

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Variable analysis

independent variables

Lipoprotein fractions in juvenile mice

dependent variables

Apolipoprotein B-containing lipoproteins VLDL and LDL elute as a single peak between fractions 14 and 18 (peak 1)
HDL-sized particles elute as a broader peak between fractions 19 and 26 (peak 2)
Total cholesterol (TC) of the fractions

control variables

Serum sample volume (100 μl per animal)
Superdex 200 gel filtration columns (10 × 300 mm; GE Healthcare Life Sciences, Chicago, IL)
Flow rate of 0.3 mL/min
Standard Tris buffer (10 mM Tris, 0.15 M sodium chloride, 1 mM EDTA, and 0.2% sodium azide)
Eluate collection as 47 1.5-mL fractions on a Frac-900 Fraction Collector (GE Healthcare) at 4°C

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