The aim of the present investigation was therefore to compare the richer culture medium DMEM/F12 to the poorer DMEM with regard to sensitivity of EndoC-H1 cells to the lipotoxic effect of sodium palmitate. Methods Cell culture Human EndoC-H1 cells were cultured in ECM/fibronectin-coated plates in low-glucose (5.5 mM) DMEM or DMEM/Ham’s F12 (50%/50%, vol/vol) with supplements as described previously.1 Palmitate (sodium salt, Sigma-Aldrich) was dissolved in 50% ethanol during heating to 60C (final concentration of ethanol: 0.50%) and was added to the 2% fatty acid free BSA (Roche) containing culture media 30 min before addition to the cell cultures. Evaluation of cell viability 105 EndoC-H1 cells were plated and pre-cultured as described above in 48-well plates for 3C5 d using either DMEM or DMEMF12 based culture media. sodium palmitate. Palmatine chloride Such an improvement would allow detailed studies on lipotoxic mechanisms using a real population of human -cells. The aim of the present investigation was therefore to compare the richer culture medium DMEM/F12 to the poorer DMEM with regard to sensitivity of EndoC-H1 cells to the lipotoxic effect of sodium palmitate. Methods Cell culture Human EndoC-H1 cells were cultured in ECM/fibronectin-coated plates in low-glucose (5.5 mM) DMEM or DMEM/Ham’s F12 (50%/50%, vol/vol) with supplements as described previously.1 Palmitate (sodium salt, Sigma-Aldrich) was dissolved in 50% ethanol during heating to 60C (final concentration of ethanol: 0.50%) and was added to the 2% fatty acid free BSA (Roche) containing culture media 30 min before addition to the cell cultures. Evaluation of cell viability 105 EndoC-H1 cells were plated and pre-cultured as described above in 48-well plates for 3C5 d using either DMEM or DMEMF12 based culture media. The cells were then cultured for various time points with or without 1.5?mM palmitate + 2% Palmatine chloride BSA. Alternatively, cells were cultured for 24?h with or without various concentrations of sodium palmitate. The cell viability of EndoC-H1 ID1 was determined by staining the cells with propidium iodide (Sigma) (5?g/ml) for 10?min at 37 C. After washing, cells were trypsinized and analyzed for red fluorescence (FL-3) using flow cytometry (FacsCalibur, BD). Insulin release Cells were preincubated for 120?min in Krebs Ringer bicarbonate buffer containing 10?mM HEPES pH 7.4, 0,1% bovine serum albumin and 2?mM glucose, and then incubated for 30?min in either 2?mM glucose or 20?mM glucose with or without 35?mM KCl or 25?M carbachol, at 37C in Krebs Ringer Bicarbonate buffer with the same additions as during the pre-incubation. Cells were then lysed in phosphate buffer saline made up of 1% Triton X-100 (Sigma Aldrich) for insulin content and total protein determinations. Insulin concentrations were measured using an Insulin Assay Kit (catalog #: 10C1113C01, Mercodia) and total cell protein by using the DC protein assay (Bio-Rad Laboratories), which is based on the Lowry assay. Results A paired comparison between culture in DMEM with DMEM/F12 exhibited a markedly increased sensitivity of EndoC-H1 cells to the apoptotic effects sodium palmitate and sodium palmitate + high glucose (Fig.?1). Time course analysis exhibited that already after a one day DMEM/F12 culture period palmitate + high glucose increased cell death markedly, and at day 2 and 3 also palmitate alone, at 5.5?mM glucose, promoted increased cell death (Fig.?1A). Using DMEM, however, palmitate + high glucose induced only a small increase in cell death at day 3. High glucose by itself did not affect cell death rates; neither in DMEM Palmatine chloride nor DMEM/F12 cultured cells. The dose response, analyzed after a 24?h palmitate exposure period, revealed that a concentration of 1 1.5?mM palmitate in DMEM/F12 medium is sufficient to promote substantial EndoC-H1 cell death, and that 22?mM of glucose potentiates the effect of palmitate (Fig.?1B). As DMEM/F12 contains linoleic acid, whereas DMEM does not, we next analyzed whether supplementation of DMEM with linoleic acid (84 g/L) mimicked the effect of DMEM/F12. Indeed, linoleic acid promoted a modest increase in cell death when co-cultured in palmitate + high glucose made up of DMEM (Fig.?1C). We also analyzed cell death in response to the cytokines interleukin-1 + IFN-, and in this case DMEM/F12 did not potentiate cell death rates as compared with DMEM (Fig.?1D). This suggests that there is no general increase in the apoptotic propensity of DMEM/F12 cultured EndoC-H1 cells as compared with cells cultured in DMEM. Open in a separate window Physique 1. EndoC-H1 cells are sensitive to sodium palmitate when cultured.