HTS using DiI-HDL to assay lipid transfer in ldlA[SR-BI] cells Measured in Cell-Based System Using Plate Reader - 2085-01_Inhibitor_Dose_CherryPick_Activity
HDL particles are labeled with DiI and added to mSR-B1 cells at 10 ug/mL in black, 384 well plates. Cells normally take up HDL via the SR-B1 scavenger receptor in 2 to 3 hours. After significant uptake of the DiI-HDL, cells become fluorescent. The level of fluorescence correlates with the amount of HDL uptake and can be measured with the Perkin Elmer Envision plate reader. The uptake can be more ..
BioActive Compounds: 65
Keywords: HDL, Scavenger receptor, SR-BI, cholesterol
HDL particles are labeled with DiI and added to mSR-B1 cells at 10 ug/mL in black, 384 well plates. Cells normally take up HDL via the SR-B1 scavenger receptor in 2 to 3 hours. After significant uptake of the DiI-HDL, cells become fluorescent. The level of fluorescence correlates with the amount of HDL uptake and can be measured with the Perkin Elmer Envision plate reader. The uptake can be inhibited with the compound BLT-1 or the fluorescence can be reduced when co-treated with an excess of unlabeled HDL. mSR-BI cells are a CHO cell line that lacks expression of the LDL receptors and overexpresses the scavenger receptor, SR-BI. mSR-BI cells are exposed to compound for 3 hours. DiI labeled HDL is added to the well at a final of 10 ug/mL. Inhibitors of SR-B1 and HDL uptake will have a reduction in fluorescence.
Plate 10,000 cells ldlA[mSR-BI] 30 ul/ well in Ham's F12/10% FCS/PSG
1) Remove media with aspirator.
2) Add 30 ul Ham's F12/0.5% BSA/25Mm HEPES pH 7.4 + 10 ug/mL DiI-HDL with Combi
3) Pin transfer 100 nl compounds and positive control (sentinel plate).
4) Incubate 3 hours @ 37 degrees C
5) Remove media with aspirator.
6) Analyze DiI-HDL uptake 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=36) and positive control wells (PC; n=36) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
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 '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: The plate pattern correction algorithm 'Runwise Pattern (Multiplicative)' in Genedata (v7.0.3) was applied to the normalized plate data.
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.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)