min6-β cells Search Results


elisa (enzyme-linked immunosorbent assay) kit for insulin detection in min6 β cells  (Mercodia Inc)
90
Mercodia Inc elisa (enzyme-linked immunosorbent assay) kit for insulin detection in min6 β cells
(A) <t>MIN6</t> <t>β</t> <t>cells</t> were incubated with palmitate (0.4 mM) for 48 h prior to incubation with glucose (3 mM or 16 mM) or with an amino acid/glucose mix (10 mM leucine and glutamine/3 mM glucose) for 1 h. Glucose-stimulated insulin secretion (GSIS) and amino acid-stimulated insulin secretion (AASIS) were then assessed as described in the . The net change in insulin secretion from 3 to 16 mM glucose or to 10 mM amino acid was analysed and graphed (B). Values are mean ± SEM from 3 separate experiments. The asterisk signifies a significant difference from the appropriate control (P<0.05).
Elisa (Enzyme Linked Immunosorbent Assay) Kit For Insulin Detection In Min6 β Cells, supplied by Mercodia Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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(A) MIN6 β cells were incubated with palmitate (0.4 mM) for 48 h prior to incubation with glucose (3 mM or 16 mM) or with an amino acid/glucose mix (10 mM leucine and glutamine/3 mM glucose) for 1 h. Glucose-stimulated insulin secretion (GSIS) and amino acid-stimulated insulin secretion (AASIS) were then assessed as described in the . The net change in insulin secretion from 3 to 16 mM glucose or to 10 mM amino acid was analysed and graphed (B). Values are mean ± SEM from 3 separate experiments. The asterisk signifies a significant difference from the appropriate control (P<0.05).

Journal: PLoS ONE

Article Title: Chronic Effects of Palmitate Overload on Nutrient-Induced Insulin Secretion and Autocrine Signalling in Pancreatic MIN6 Beta Cells

doi: 10.1371/journal.pone.0025975

Figure Lengend Snippet: (A) MIN6 β cells were incubated with palmitate (0.4 mM) for 48 h prior to incubation with glucose (3 mM or 16 mM) or with an amino acid/glucose mix (10 mM leucine and glutamine/3 mM glucose) for 1 h. Glucose-stimulated insulin secretion (GSIS) and amino acid-stimulated insulin secretion (AASIS) were then assessed as described in the . The net change in insulin secretion from 3 to 16 mM glucose or to 10 mM amino acid was analysed and graphed (B). Values are mean ± SEM from 3 separate experiments. The asterisk signifies a significant difference from the appropriate control (P<0.05).

Article Snippet: The ELISA (Enzyme-Linked ImmunoSorbent Assay) kit for insulin detection in MIN6 β cells was from Mercodia (Uppsala, Sweden).

Techniques: Incubation

MIN6 β cells were incubated in the absence or presence of palmitate (0.4 mM) for 48 h prior to analysis of cell number by DAPI staining (A) or (B) caspase 3/7 activity (a proapoptotic marker). Three random visual fields from duplicate samples were chosen (typical phase, DAPI stained and merged images are shown in panel A), and adherent cell number noted from control or palmitate-treated cells following DAPI staining (A and B). Alternatively, following the fatty acid incubation period cells were incubated with caspase 3/7 assay reagent and caspase 3/7 activity determined by luminescence as described in (B). In some experiments, cells that had been pre-treated with palmitate (0.4 mM, 48 h) were subsequently incubated with glucose (16 mM) or with an amino acid/glucose mix (10 mM leucine and glutamine/3 mM glucose) for 1 h prior to analysis of cell number by DAPI staining (C). Bar values are mean ± SEM from 3 or 4 separate experiments. The asterisk signifies a significant difference from the appropriate control value (P<0.05).

Journal: PLoS ONE

Article Title: Chronic Effects of Palmitate Overload on Nutrient-Induced Insulin Secretion and Autocrine Signalling in Pancreatic MIN6 Beta Cells

doi: 10.1371/journal.pone.0025975

Figure Lengend Snippet: MIN6 β cells were incubated in the absence or presence of palmitate (0.4 mM) for 48 h prior to analysis of cell number by DAPI staining (A) or (B) caspase 3/7 activity (a proapoptotic marker). Three random visual fields from duplicate samples were chosen (typical phase, DAPI stained and merged images are shown in panel A), and adherent cell number noted from control or palmitate-treated cells following DAPI staining (A and B). Alternatively, following the fatty acid incubation period cells were incubated with caspase 3/7 assay reagent and caspase 3/7 activity determined by luminescence as described in (B). In some experiments, cells that had been pre-treated with palmitate (0.4 mM, 48 h) were subsequently incubated with glucose (16 mM) or with an amino acid/glucose mix (10 mM leucine and glutamine/3 mM glucose) for 1 h prior to analysis of cell number by DAPI staining (C). Bar values are mean ± SEM from 3 or 4 separate experiments. The asterisk signifies a significant difference from the appropriate control value (P<0.05).

Article Snippet: The ELISA (Enzyme-Linked ImmunoSorbent Assay) kit for insulin detection in MIN6 β cells was from Mercodia (Uppsala, Sweden).

Techniques: Incubation, Staining, Activity Assay, Marker

MIN6 β cells were incubated either with or without 10 µM AZ422 (a SPT inhibitor) or 5 µM fenretinide (an inhibitor of dihydroceramide desaturase) (A) for 48 h or with varying concentrations of ceramide and dihydroceramide (B–F) for 2 h. Palmitate (0.4 mM) was present for 48 h where indicated. Glucose was added for 1 h for analysis of GSIS (A and B). Alternatively, cells were either lysed for immunoblot analysis using antibodies against (C) PKCζ/λ Thr410 and total PKCζ/λ (used as loading control), (D) PKB Ser473 and total PKB (as a loading control) or harvested and lipids extracted to assess (E) ceramide or (F) DAG content. Bars represent mean ± SEM of 3 experiments and asterisks signify a significant difference from the untreated control value (P<0.05).

Journal: PLoS ONE

Article Title: Chronic Effects of Palmitate Overload on Nutrient-Induced Insulin Secretion and Autocrine Signalling in Pancreatic MIN6 Beta Cells

doi: 10.1371/journal.pone.0025975

Figure Lengend Snippet: MIN6 β cells were incubated either with or without 10 µM AZ422 (a SPT inhibitor) or 5 µM fenretinide (an inhibitor of dihydroceramide desaturase) (A) for 48 h or with varying concentrations of ceramide and dihydroceramide (B–F) for 2 h. Palmitate (0.4 mM) was present for 48 h where indicated. Glucose was added for 1 h for analysis of GSIS (A and B). Alternatively, cells were either lysed for immunoblot analysis using antibodies against (C) PKCζ/λ Thr410 and total PKCζ/λ (used as loading control), (D) PKB Ser473 and total PKB (as a loading control) or harvested and lipids extracted to assess (E) ceramide or (F) DAG content. Bars represent mean ± SEM of 3 experiments and asterisks signify a significant difference from the untreated control value (P<0.05).

Article Snippet: The ELISA (Enzyme-Linked ImmunoSorbent Assay) kit for insulin detection in MIN6 β cells was from Mercodia (Uppsala, Sweden).

Techniques: Incubation, Western Blot

MIN6 β cells were incubated in the absence or presence of palmitate (0.4 mM) for 48 h. Glucose (16 mM) was added for 1 h and insulin (100 nM) in the penultimate 10 min of this incubation period. Cells were harvested and immunoblotted for phospho-PKB Ser473 (A) and phospho-ERK Thr202/Tyr204 (B) and normalised against total protein. Bars represent mean ± SEM from 3 separate experiments and asterisks signify a significant difference from the untreated control values (P<0.05).

Journal: PLoS ONE

Article Title: Chronic Effects of Palmitate Overload on Nutrient-Induced Insulin Secretion and Autocrine Signalling in Pancreatic MIN6 Beta Cells

doi: 10.1371/journal.pone.0025975

Figure Lengend Snippet: MIN6 β cells were incubated in the absence or presence of palmitate (0.4 mM) for 48 h. Glucose (16 mM) was added for 1 h and insulin (100 nM) in the penultimate 10 min of this incubation period. Cells were harvested and immunoblotted for phospho-PKB Ser473 (A) and phospho-ERK Thr202/Tyr204 (B) and normalised against total protein. Bars represent mean ± SEM from 3 separate experiments and asterisks signify a significant difference from the untreated control values (P<0.05).

Article Snippet: The ELISA (Enzyme-Linked ImmunoSorbent Assay) kit for insulin detection in MIN6 β cells was from Mercodia (Uppsala, Sweden).

Techniques: Incubation

MIN6 β cells were incubated in the absence or presence of 20 µM U0126, as indicated prior to being exposed to either 3 mM or 16 mM glucose for 1 h or alternatively to an amino acid stimulus (10 mM leucine/glutamine and 3 mM glucose). At the end of this incubation cells were (A) lysed for analysis of ERK phosphorylation in whole cell lysates or (B) prepared for analysis of net changes in GSIS or AASIS (B) as described in . Bars represent mean ± SEM from 3 separate experiments and asterisks signify a significant difference from the untreated control values (P<0.05).

Journal: PLoS ONE

Article Title: Chronic Effects of Palmitate Overload on Nutrient-Induced Insulin Secretion and Autocrine Signalling in Pancreatic MIN6 Beta Cells

doi: 10.1371/journal.pone.0025975

Figure Lengend Snippet: MIN6 β cells were incubated in the absence or presence of 20 µM U0126, as indicated prior to being exposed to either 3 mM or 16 mM glucose for 1 h or alternatively to an amino acid stimulus (10 mM leucine/glutamine and 3 mM glucose). At the end of this incubation cells were (A) lysed for analysis of ERK phosphorylation in whole cell lysates or (B) prepared for analysis of net changes in GSIS or AASIS (B) as described in . Bars represent mean ± SEM from 3 separate experiments and asterisks signify a significant difference from the untreated control values (P<0.05).

Article Snippet: The ELISA (Enzyme-Linked ImmunoSorbent Assay) kit for insulin detection in MIN6 β cells was from Mercodia (Uppsala, Sweden).

Techniques: Incubation

MIN6 β cells were incubated with 10 µM KN62 (an inhibitor of CaMKII) for 2 h. In the last hour of this incubation cells were incubated with either glucose (16 mM) or amino acids (10 mM) prior to assessing (A) GSIS or AASIS, (B) ERK activation by analysis of ERK Thr202/Tyr204 phosphorylation. Total ERK content was used for assaying protein loading. Alternatively, MIN6 β cells were incubated in the absence or presence of palmitate (0.4 mM) for 48 h and then stimulated insulin secretion assessed by the addition of glucose or amino acids for 1 h at the concentrations indicated. Cells were lysed and lysates immunoblotted to assay phospho-CaMKII Thr286 phosphorylation with native CaMKII serving as a gel loading control (C). Bars represent mean ± SEM from 3 separate experiments and asterisks signify a significant difference from the untreated control values (P<0.05).

Journal: PLoS ONE

Article Title: Chronic Effects of Palmitate Overload on Nutrient-Induced Insulin Secretion and Autocrine Signalling in Pancreatic MIN6 Beta Cells

doi: 10.1371/journal.pone.0025975

Figure Lengend Snippet: MIN6 β cells were incubated with 10 µM KN62 (an inhibitor of CaMKII) for 2 h. In the last hour of this incubation cells were incubated with either glucose (16 mM) or amino acids (10 mM) prior to assessing (A) GSIS or AASIS, (B) ERK activation by analysis of ERK Thr202/Tyr204 phosphorylation. Total ERK content was used for assaying protein loading. Alternatively, MIN6 β cells were incubated in the absence or presence of palmitate (0.4 mM) for 48 h and then stimulated insulin secretion assessed by the addition of glucose or amino acids for 1 h at the concentrations indicated. Cells were lysed and lysates immunoblotted to assay phospho-CaMKII Thr286 phosphorylation with native CaMKII serving as a gel loading control (C). Bars represent mean ± SEM from 3 separate experiments and asterisks signify a significant difference from the untreated control values (P<0.05).

Article Snippet: The ELISA (Enzyme-Linked ImmunoSorbent Assay) kit for insulin detection in MIN6 β cells was from Mercodia (Uppsala, Sweden).

Techniques: Incubation, Activation Assay

(A) MIN6 β cells were incubated with varying concentrations of palmitate and 2-bromopalmitate (48 h) as indicated. Glucose (16 mM) was added for 1 h during serum starvation and the net change in glucose-stimulated insulin secretion assessed. (B) Cells were incubated in the absence or presence of 0.4 mM 2-bromopalmitate for 48 h and with glucose at the indicated concentration for 1 h. Cells were lysed and lysates immunoblotted for analysis of ERK Thr202/Tyr204 , PKB Ser473 , CaMKII Thr286 and total PKB, ERK and CaMKII. (C) Cells were incubated in the absence or presence of palmitate (0.4 mM) for 48 h and then after serum starvation cells were exposed to either L-carnitine (1 mM) or etomoxir (0.2 mM) for 2 h as indicated. Glucose (16 mM) was added during the final hour to induce GSIS and media removed for analysis of insulin content. Bars represent mean ± SEM from 3 separate experiments and asterisks or a cross signify a significant difference from the untreated control values or between indicated bars (P<0.05).

Journal: PLoS ONE

Article Title: Chronic Effects of Palmitate Overload on Nutrient-Induced Insulin Secretion and Autocrine Signalling in Pancreatic MIN6 Beta Cells

doi: 10.1371/journal.pone.0025975

Figure Lengend Snippet: (A) MIN6 β cells were incubated with varying concentrations of palmitate and 2-bromopalmitate (48 h) as indicated. Glucose (16 mM) was added for 1 h during serum starvation and the net change in glucose-stimulated insulin secretion assessed. (B) Cells were incubated in the absence or presence of 0.4 mM 2-bromopalmitate for 48 h and with glucose at the indicated concentration for 1 h. Cells were lysed and lysates immunoblotted for analysis of ERK Thr202/Tyr204 , PKB Ser473 , CaMKII Thr286 and total PKB, ERK and CaMKII. (C) Cells were incubated in the absence or presence of palmitate (0.4 mM) for 48 h and then after serum starvation cells were exposed to either L-carnitine (1 mM) or etomoxir (0.2 mM) for 2 h as indicated. Glucose (16 mM) was added during the final hour to induce GSIS and media removed for analysis of insulin content. Bars represent mean ± SEM from 3 separate experiments and asterisks or a cross signify a significant difference from the untreated control values or between indicated bars (P<0.05).

Article Snippet: The ELISA (Enzyme-Linked ImmunoSorbent Assay) kit for insulin detection in MIN6 β cells was from Mercodia (Uppsala, Sweden).

Techniques: Incubation, Concentration Assay