Primary beta islet insulin ELISA Measured in Cell-Based System Using Plate Reader - 2061-09_Inhibitor_Dose_DryPowder_Activity
This assay measures glucose-induced insulin secretion, the gold standard for beta-cell function. The insulin stimulatory index can be measured by ELISA, after 1-hour incubation with "high glucose" (typically 16.7 mM) in the experimental buffer, in comparison with "low glucose" (typically 1.67 mM). The rat INS-1E cell line is derived from a clone selected for high levels of insulin secretion in more ..
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Keywords: insulin, beta cell, ELISA, primary human pancreatic beta islets
This assay measures glucose-induced insulin secretion, the gold standard for beta-cell function. The insulin stimulatory index can be measured by ELISA, after 1-hour incubation with "high glucose" (typically 16.7 mM) in the experimental buffer, in comparison with "low glucose" (typically 1.67 mM). The rat INS-1E cell line is derived from a clone selected for high levels of insulin secretion in response to glucose, and thus serves as a good model of beta-cell function. Beta cells treated with cytokines lose their insulin secretory response to glucose. Small molecules that can promote beta-cell survival should restore insulin secretion.
Cell and Human Islet Culture. 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 96-well plates containing extracellular matrix secreted by the HTB-9 human bladder carcinoma cell line.
Primary human islets were incubated for 6 days in 100 uL of fresh RPMI containing 1% FBS and the cytokine cocktail, in the presence or absence of compounds. Media was refreshed after 3 days. Cells were washed and incubated for 2 h in KRBH (135 mM NaCl, 3.6 mM KCl, 5 mM NaHCO3, 0.5 mM NaH2PO4, 0.5 mM MgCl2, 1.5 mM CaCl2, 10 mM HEPES, pH 7.4, 0.1% BSA) without glucose. Cells were subsequently incubated with KRBH containing 2 or 15 mM glucose for 1 hour. The supernatant was taken for measurement of released insulin. Insulin was measured with a rat insulin ELISA kit (Alpco).
ALPCO INSULIN ELISA PROTOCOL
Bring all reagents and microplate strips to room temperature prior to use. Gently mix all reagents before use. A standard curve must be performed with each assay and with each microplate if more than one is run at a time. All standards, samples, and the control should be run in duplicate.
1. Ensure that microplates are at room temperature prior to opening foil pouch. Designate enough microplate strips for the standards, desired number of samples, and control. The remaining strips should be stored at 2-8 deg C in the tightly sealed foil pouch containing the desiccant.
2. Pipette 5 ul of each standard, reconstituted control (see Reagent Preparation), or sample into its respective wells.
3. Pipette 75 ul of Working Strength Conjugate (see Reagent Preparation) into each well.
4. Incubate for 2 hours, shaking at 700-900 rpm on a horizontal microplate shaker at room temperature (18-25 deg C).
5. Wash the microplate 6 times with Working Strength Wash Buffer (see Reagent Preparation) with a microplate washer. Alternatively, use a wash bottle to fill the wells, and then discard the liquid, inverting and firmly tapping the microplate on absorbent paper towels between washes. After the final wash with either the microplate washer or wsah bottle, remove any residual Wash Buffer and bubbles from the wells by inverting and firmly tapping the microplate on absorbent paper towels (See Microplate Locking Diagram below).
6. Pipette 100 ul of TMB Substrate into each well.
7. Incubate for 15 minutes at room temperature (18-25 deg C) on a horizontal microplate shaker (700-900 rpm).
8. Pipette 100 ul of Stop Solution into each well. Gently shake the microplate to stop the reaction. Remove bubbles before reading with microplate reader.
9. Place the microplate in a microplate reader capable of reading the absorbance at 450nm with a reference wavelength of 620-650nm. The microplate should be analyzed within 30 minutes following the addition of Stop Solution.
PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate. The positive control condition was absence of cytokine.
EXPECTED OUTCOME: Active compounds result in increasing 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).
A standard curve run was run on each assay plate using reference material provided in the ELISA kit. Background-subtracted absorbance values were calculated for each well as (Abs450 - Abs620), and used to extrapolate quantity of insulin secreted in micro International Units (uIU) from the standard curve. No further data normalization methods were applied.
PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.
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 >= 30% but <= 70%, 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.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)