Apoptosis Measured in Cell-Based System Using Plate Reader - 2061-03_Inhibitor_Dose_DryPowder_Activity_Set2
In apoptosis, electron transport is disrupted, cytochrome c is released, and the mitochondroal membrane and the electrochemical potential across the mitochondrial membrane becomes reduced. In a normal, mitochondria, JC-1 is a cationic dye which exhibits a membrane potential-dependent accumulation in the mitochondrion. The dye exists as a monomer at low concentrations and yields green more ..
BioActive Compounds: 15
Keywords: Mitochondra, INS-1E, type I diabetes, JC-1
In apoptosis, electron transport is disrupted, cytochrome c is released, and the mitochondroal membrane and the electrochemical potential across the mitochondrial membrane becomes reduced. In a normal, mitochondria, JC-1 is a cationic dye which exhibits a membrane potential-dependent accumulation in the mitochondrion. The dye exists as a monomer at low concentrations and yields green fluorescence, similar to fluorescein. At higher concentrations, the dye forms J-aggregates that exhibit a broad excitation spectrum and an emission maximum at ~590 nm (i.e. red fluorescence). In apoptotic cells, JC-1 remains in the cytoplasm as the green monomer. Mitochondrial depolarization is indicated by a decrease in the red to green intensity ratio. The color shift is attributed to the concentration dependant accumulation of red fluorescent J-aggregates. In this assay, INS-1E insulinoma cells will be treated with 10 ng/mL IL-1 beta, 50 ng/mL IFN-gamma, 25 ng/mL TNF-alpha for 48 hours in the presence of several doses of compounds.
1. Seed cells into black 384-well optical bottom plates at 8000 cells per well using 50mL/well with Combi.
2. Change media to 50uL/well prior to compound addition (usually on day 2). After overnight incubation, medium was removed and 50uL RPMI containing 1% FBS and a combination of cytokines (10ng/mL IL-1b, 50ng/mL IFN-g, 25ng/mL TNF-a) was added to every well. Using libraries of compounds dissolved in DMSO and a CyBi-Well pin-transfer robot (CyBio Corp.), 0.1 uL of each compound was added to the wells.
3. After incubation for 48 hr, medium was removed. Dilute 1mM JC-1 stock in phenol-red free media to a final concentration of 3.25mM. Aspirate plates, and add 20mL/well.
4. Incubate plates at 37 deg C for 2 hours.
5. Wash cells three times with 50ul/well of 1X PBS.
6. Read fluorescence on the Envision plate reader using the rhodamine channel (ex/em 530/580) followed by the fluorescein channel (ex/em 485nm/530nm). Each time a new assay is read, run an optimization cycle on the instrument to ensure the appropriate reading levels for the assay.
PRESENCE OF CONTROLS: Neutral control wells (NC) were included on every plate.
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).
The raw signals of the plate wells were normalized using the 'Neutral Controls' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral controls (NC) is set to a normalized activity value of 0.
A normalized activity value of 100 is defined as (2)(NC).
A normalized activity value of -50 is defined as (0.5)(NC).
Experimental wells values were scaled to this range.
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.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)