|HDL Counter Assay: 3/24 Cytotox Measured in Cell-Based System Using Plate Reader - 2085-02_Inhibitor_Dose_DryPowder_Activity_Set4 - BioAssay Summary
The HDL uptake primary assay is looking for a decrease in fluorescence. It is possible that a compound will cause cells to decrease HDL uptake due to non-specific consequences of cellular toxicity. Therefore, we would like to make sure that lead compounds are not toxic in the 3 hour time period used in the primary assay. Cells are treated with compounds for 24 hours and then cell viability is more ..
BioActive Compounds: 8
Depositor Specified Assays
Keywords: cytotoxicity, Cell titer glo, ldlA[SRB1] cells
The HDL uptake primary assay is looking for a decrease in fluorescence. It is possible that a compound will cause cells to decrease HDL uptake due to non-specific consequences of cellular toxicity. Therefore, we would like to make sure that lead compounds are not toxic in the 3 hour time period used in the primary assay. Cells are treated with compounds for 24 hours and then cell viability is measured using the Cell Titer Glo Assay which is a luciferase-based reagent that measures cellular ATP levels. Compounds will be tested at 8 different doses to determine IC50 values. Compounds that are toxic after 3 hours at an IC50 less than 30 uM will be excluded from additional studies. Compounds that are active in the primary assay but toxic below 30 uM at 24 hours will still be pursued.
Compounds that are cytotoxic will decrease cellular ATP levels resulting in a decrease in luminescence.
Plate 4,000 cells ldlA[mSR-BI] 30 microl/well in Ham's F12/10% FCS/PSG
1)Pin transfer 100 nl compounds and positive control (sentinel plate).
2)Incubate 24 hours @ 37 degrees C
3)Add 20 uL Promega Cell titer glo per well
4)Shake plate for 15 seconds
5)Incubate 10 minutes
6)Read luminescence with Perkin-Elmer Envision plate reader
PRESENCE OF CONTROLS: Neutral control wells (NC; n=104) and positive control wells (PC; n=20) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
The compounds were assayed in multiple independent instances using an identical protocol; each instance is called a 'test'. For each test, the following analysis was applied:
ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal (2)(Max_Concentration).
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
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.
pAC was set to equal -1*log10(AC)
Activity_Outcome = 1 (inactive) when:
a) 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
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
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 deemed not valid due to poor fit quality.
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.
Once the data for each test was processed, the test number was appended to all column headers in that test's data set. The individual test results were then aggregated as follows:
1. The final PUBCHEM_ACTIVITY_OUTCOME was set to:
a. '2' (active) when all test outcomes were '2' (active), or
b. '1' (inactive) when all test outcomes were '1' (inactive), or
c. '3' (inconclusive) when the test outcomes were mixed.
2. The final ACTIVE_CONCENTRATION (AC) was set as follows:
a. If the final PUBCHEM_ACTIVITY_OUTCOME = 2, AC was set as the mean of the constituent test active concentrations;
b. If the final PUBCHEM_ACTIVITY_OUTCOME = 1 or 3, AC was left empty.
3. The final PUBCHEM_ACTIVITY_SCORE was calculated based on the aggregated ACTIVE_CONCENTRATION, using the same logic described above for individual test scores.
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)