Metabolomic Analysis of Retinal and RPE Cells

Isolated mouse retina or confluent human fetal RPE cells were changed into pre-warmed Krebs-Ringer bicarbonate buffer (KRB) containing, depending on the experiment, [1,2] ¹³C glucose, U-¹³C glucose, or U-¹³C lactate (Sigma) as described elsewhere (Du et al., 2013a (link); Du et al., 2015 (link); Du et al., 2016b (link)). Both retinas and RPE cells were incubated for the specified time points. Metabolites from each time point were extracted and analyzed by gas chromatography mass spectrometry (GC-MS, Agilent 7890/5975C) as described in detail (Du et al., 2013a (link); Du et al., 2013b (link)).

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Kanow M.A., Giarmarco M.M., Jankowski C.S., Tsantilas K., Engel A.L., Du J., Linton J.D., Farnsworth C.C., Sloat S.R., Rountree A., Sweet I.R., Lindsay K.J., Parker E.D., Brockerhoff S.E., Sadilek M., Chao J.R, & Hurley J.B. (2017). Biochemical adaptations of the retina and retinal pigment epithelium support a metabolic ecosystem in the vertebrate eye. eLife, 6, e28899.

Publication 2017

U-13C glucose Agilent 7890/5975C

Bicarbonate Buffer Cells Fetal Gc ms Glucose Human Lactate Mouse Retinas

Corresponding Organization :

Other organizations : University of Washington, Seattle University, West Virginia University, Fred Hutch Cancer Center

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Protocol cited in 3 other protocols

Variable analysis

independent variables

[1,2] 13C glucose
U-13C glucose
U-13C lactate

dependent variables

Metabolites

control variables

Isolated mouse retina
Confluent human fetal RPE cells
Krebs-Ringer bicarbonate buffer (KRB)
Incubation time points

controls

No positive or negative controls are explicitly mentioned.

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