Extracellular Matrix Secretion Protocol

HCFs, T1DMs, and T2DMs were seeded at a density of 1 × 10⁶/well on 0.4 μM polycarbonate membranes (Corning, Corning, NY, USA), as previously described [27 (link), 30 (link)–32 (link)]. Cultures were maintained in EMEM, 10% FBS, and 1% AA and further stimulated by 0.5 mM stable vitamin C (VitC; 0.5 mM 2-O-α-D-glucopyranosyl-L-ascorbic acid, Sigma-Aldrich, St. Louis, MO, USA) in order to promote secretion and assembly of extracellular matrix (ECM). The constructs were maintained in VitC media for 2 or 4 weeks with media changes occurring every other day [29 (link)].

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Shrestha P., Whelchel A.E., Nicholas S.E., Liang W., Ma J.X, & Karamichos D. (2022). Monocarboxylate Transporters: Role and Regulation in Corneal Diabetes. Analytical Cellular Pathology (Amsterdam), 2022, 6718566.

Publication 2022

0.4 μM polycarbonate membranes 2-O-α-D-glucopyranosyl-L-ascorbic acid

Extracellular matrix Membranes Polycarbonate Secretion Vitc

Corresponding Organization : University of North Texas Health Science Center

Other organizations : University of Oklahoma Health Sciences Center, Wake Forest University

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Variable analysis

independent variables

Seeding density (1 × 10^6/well)
Polycarbonate membrane (0.4 μM)
Stimulation with vitamin C (0.5 mM 2-O-α-D-glucopyranosyl-L-ascorbic acid)

dependent variables

Secretion and assembly of extracellular matrix (ECM)

control variables

Cell types (HCFs, T1DMs, T2DMs)
Culture medium (EMEM, 10% FBS, 1% AA)
Culture duration (2 or 4 weeks)

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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