Primary Beta Cell Apoptosis assay Measured in Cell-Based System Using Plate Reader - 2061-07_Inhibitor_Dose_DryPowder_Activity_Set2
The best indication of physiological relevance in cell culture is to recapitulate our results in human beta cells. Therefore, we will test the effects of promising screening positives in primary human pancreatic islets. We have access to human islets through the Juvenile Diabetes Research Foundation, and have confirmed that dissociated human islet cells are sensitive to cytokine treatment. more ..
BioActive Compound: 1
Keywords: ELISA, insulin, apoptosis, primary human beta cells
The best indication of physiological relevance in cell culture is to recapitulate our results in human beta cells. Therefore, we will test the effects of promising screening positives in primary human pancreatic islets. We have access to human islets through the Juvenile Diabetes Research Foundation, and have confirmed that dissociated human islet cells are sensitive to cytokine treatment. Primary human pancreatic islets containing beta cells are cultured in the presence of cytokines (IFN, TNF and IL1Beta) for 3 and 6 days. They are exposed to high glucose to stimulate insulin production. Supernatants are collected for the insulin ELISA while cells are tested for apoptotic activity with the Caspase-glo 3/7 assay (Promega). Compounds that protect beta cells as measured by restoration of insuliation secretion will be suitable for probe declaration.
Human islets were obtained through the Islet Cell Resource Consortium (http://icr.coh.org/) and through the National Disease Research Interchange (http://www.ndriresource.org/). The purity and viability of human islets are reported to be 70-93% and 70-98%, respectively, and the average age of cadaveric donors was 40.7 +/- 9.0 y (range 32-57 y; n = 6). Islets were washed with PBS and incubated in CMRL medium supplemented with 10% FBS, 2 mM glutamine, 100 U/mL penicillin, and 100 ug/mL streptomycin. Islets were gently dissociated into a cell suspension by incubating in Accutase (37 deg C, 10 min), and seeded in 384-well plates containing extracellular matrix secreted by the HTB-9 human bladder carcinoma cell line.
Primary human islets were incubated for 6 days in 50 uL of fresh RPMI containing 1% FBS and the cytokine cocktail, in the presence or absence of compounds. Media was changed after 3 days. After 6 days, an equal volume of Caspase-glo 3/7 (Promega) was added per well and incubated at room temperature for 2 hours. After 2 hours, luminescence was measured with the Perkin-Elmer Envison plate reader.
PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.
NC wells contained DMSO only (no compound).
PC wells contained DMSO only and no cytokine.
Plate well values (W) were normalized according to this formula: ((W - NC_mean) / (PC_mean - NC_mean))
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (HVC) was determined and the active concentration limit (AC_limit) was set to equal (10)(HVC).
MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION(AC): AC50
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.
Activity_Outcome = 1 (inactive) when:
a) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
b) AC > AC_limit, or
c) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null
Activity_Outcome = 2 (active) when:
AC <= AC_limit
Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= -70% but <= -30%, or
b) The fit was not valid due to poor fit quality.
PUBCHEM_ACTIVITY_SCORE = 0 when PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive)
PUBCHEM_ACTIVITY_SCORE = (-10)[Log(AC)], where AC is in molar, when PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
PUBCHEM_ACTIVITY_SCORE = 100 when the curve fit is constant and showing full inhibition at all tested concentrations.
The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.
All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.
Categorized Comment - additional comments and annotations
Assay Format: Cell-based
Assay Cell Type: Beta cell
Assay Format: Cell-based
Assay Type: Functional
* Activity Concentration. ** Test Concentration.
Data Table (Concise)