DiI Quencher Assay Measured in Biochemical System Using Plate Reader - 2085-08_Inhibitor_Dose_DryPowder_Activity
Assay Overview: The primary assay for the HDL project leads to a reduction in fluorescence by potential inhibitors. One possible outcome is that the compounds have inherent fluorescence and reduce signal in a dose-dependent manner without actually altering any biology. We created an assay where compounds are pinned into assay buffer containing DiI-HDL without any cells. Compounds are incubated with DiI-HDL at 10 ug/mL in Ham's/HEPES/BSA media for 30 minutes and then assay plates are measured with the identical settings for the primary assay. ..more
Keywords: fluorescence quenching, DiI, cell -free
Assay Overview: The primary assay for the HDL project leads to a reduction in fluorescence by potential inhibitors. One possible outcome is that the compounds have inherent fluorescence and reduce signal in a dose-dependent manner without actually altering any biology. We created an assay where compounds are pinned into assay buffer containing DiI-HDL without any cells. Compounds are incubated with DiI-HDL at 10 ug/mL in Ham's/HEPES/BSA media for 30 minutes and then assay plates are measured with the identical settings for the primary assay.
Expected Outcome: Compounds that quench DiI fluorescence will lead to a dose dependent loss of signal in this assay. Any compound that does not alter fluorescence and has an IC50 of >30 uM will be considered for further studies.
1)#Add 30microl Ham's F12/0.5% BSA/25 mM HEPES pH 7.4 + 10 microg/mL DiI-HDL with Combi
2)#Pin transfer 100 nl compounds and positive control.
3)#Incubate 30 minutes @ room temperature
4)#Analyze DiI-HDL signal with 'Envision' Bodipy TMR mirror #405, Excitation filter is Photometric 550 (#312) and emission filter is Cy3 595 (#229) with bottom read
PRESENCE OF CONTROLS: Neutral control wells (NC; n=32) were included on every plate.
EXPECTED OUTCOME: Active quenching compounds result in decreasing readout signal. Since, we are testing a loss of DiI fluorescence in the primary assay, an inactive compound is the desired outcome.
ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was 35 uM.
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.
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.
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)