HDL Orthogonal Assay: Alexa 488-HDL Measured in Cell-Based System Using Plate Reader - 2085-03_Inhibitor_Dose_DryPowder_Activity
HDL binding is assessed using Alexa488-HDL. The assay is similar to the primary DiI-HDL screening assay, except Alexa488-HDL will be used in place of DiI. DiI non-specifically enters lipid membranes both on HDL particles and cell membranes. For this assay, the Alexa488 is directly bound to components of the HDL particle and so no transfer of the fluorophore to cell membranes occurs. Instead, direct binding of the HDL particles to the SR-BI receptor can be measured. Compounds will be tested at dose. ..more
BioActive Compounds: 53
Keywords: HDL uptake, SR-BI,
HDL binding is assessed using Alexa488-HDL. The assay is similar to the primary DiI-HDL screening assay, except Alexa488-HDL will be used in place of DiI. DiI non-specifically enters lipid membranes both on HDL particles and cell membranes. For this assay, the Alexa488 is directly bound to components of the HDL particle and so no transfer of the fluorophore to cell membranes occurs. Instead, direct binding of the HDL particles to the SR-BI receptor can be measured. Compounds will be tested at dose.
Expected Outcome: It is possible that a compound can reduce binding of HDL to the receptor and this would lead to a decrease in signal. To date, most compounds, like BLT-1, seem to increase binding to the receptor leading to an increase in signal. This assay is used to decipher the mechanism of activity for a particular compound. Therefore, any outcome in the assay is acceptable.
Plate 10,000 cells ldlA[mSR-BI] 30microl/ well in Ham's F12/10% FCS/PSG
1)Remove media with aspirator.
2)Add 30microl Ham's F12/0.5% BSA/25Mm HEPES pH 7.4 + 10 microg/mL Alexa-488-HDL with Combi
3)Pin transfer 100 nl compounds and positive control.
4)Incubate 3 hours @ 37 degrees C
5)Remove media with aspirator.
6)Rinse cells with PBS (+Ca2+/Mg2+)
8)Analyze Alex-488-HDL uptake with 'Envision' with bottom read
PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.
EXPECTED OUTCOME: Compounds causing either an increase or a decrease in signal were considered active.
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 (10)(Max_Concentration).
The raw signals of the plate wells were normalized using the 'Stimulators Minus Neutral Controls' 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.
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)