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ZenComplete™: Human Islet Assays

The global prevalence of diabetes mellitus has reached epidemic proportions and is projected to continue to rise for the foreseeable future. It is estimated that diabetes will be the 7th leading cause of death worldwide by 2030. Both major forms of diabetes mellitus, type 1 (T1D) and type 2 (T2D), are ultimately caused by a failure of pancreatic β-cells to deliver insulin at a rate sufficient to account for metabolic demand. The decline of β-cell function is attributed to either decreased insulin secretion or loss of β-cell mass, lending to loss of glycemic control. Compounds that increase insulin secretion and cell proliferation or decrease cell death may be considered as therapeutic strategies for diabetes. Our primary human islet assays encompass glucose-stimulated insulin secretion (GSIS) measurement in intact islets (Fig. 1), cell proliferation measurement in dispersed islets (Fig. 2) and cell death measurement in dispersed islets (Fig. 3).

GSIS

GSIS can be measured after acute or chronic treatment of islets with compounds. Additional glucose concentrations can also be measured.

GSIS Figure 1
Figure 1. Intact primary human islets were plated on HTB9-coated 96-well plates and cultured for 72 h before GSIS assay. For the GSIS assay, islets were cultured in low glucose KRB (1.67 mM) or high glucose KRB (16.7 mM) for 2 h +/- 50 nM Ex4. Conditioned media was collected to measure secreted insulin via ELISA. Islets were then lysed, and lysates were collected to measure total insulin via ELISA. Data represent the ratio of secreted insulin to total insulin.

Islet Cell Proliferation

Cell proliferation can be measured after acute or chronic treatment of islets with compounds. Total islet cell proliferation or cell type-specific proliferation is measured by combining ClickIT technology with insulin and glucagon antibody staining to recognize beta cell-specific or alpha cell-specific proliferation, respectively. The measurement is conducted in a 96-well format with Ad-Cyclin D1 as the positive control and Ad-GFP as the negative control.

Islet Cell Proliferation Figure 2
Figure 2. Primary human islets were dispersed, plated on HTB9-coated 96-well plates and allowed to attach. Medium containing adenovirus was added for 18 h and then replaced with medium containing EdU. Islets were cultured for 72 h, and EdU incorporation was measured via ClickIT technology and high-content imaging. Data represent the percent EdU incorporation of all cells.

Islet Cell Death

Cell death can be measured after acute or chronic treatment of islets with compounds. Total islet cell or cell type-specific death is measured in this assay by utilizing insulin and glucagon antibodies. ImageIT staining is used to measure both necrosis and apoptosis, and TUNEL staining is used to measure only apoptosis. The measurement is conducted in a 96-well format with 10 mM streptozotocin (STZ) as the positive control and untreated cells as the negative control.

Islet Cell Death Figure 3
Figure 3. Primary human islets were dispersed, plated on HTB9-coated 96-well plates and allowed to attach. Medium containing STZ was added for 48 h. Beta cell-specific death was measured via co-staining of ImageIT and insulin (INS) antibodies, and alpha cell-specific death was measured via co-staining of ImageIT and glucagon (GCG) antibodies. Cells were imaged by a high-content imager.

Ordering Information

ZenComplete™: Human Islet Assays

Item#Item DescNoteU/MPrice
CA-73GSIS Assay, Human Islets96-well plate AssayContact US
CA-71Proliferation Assay-Human Islets96-well plate AssayContact US
Prices for contract services vary depending on number of sample numbers, special conditions, compound solubility, etc. Minimum charges will apply. Please contact us for detailed information and a price quote on your project.

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