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_DryPowder_Activity_Set9
The goal of this assay is to identify compounds that disrupt the uptake of HDL particles (Inhibitors) to scavenger receptor class B, type I (SR-BI) or increase uptake (Activators). To measure this binding event, HDL particles are labeled with the fluorescent dye DiI and added to mSR-BI cells. Cells take up HDL via SR-BI in 2 to 3 hours. After significant uptake of the DiI-HDL, cells become more ..
BioActive Compounds: 19
Keywords: HDL, Scavenger receptor, SR-BI, cholesterol
The goal of this assay is to identify compounds that disrupt the uptake of HDL particles (Inhibitors) to scavenger receptor class B, type I (SR-BI) or increase uptake (Activators). To measure this binding event, HDL particles are labeled with the fluorescent dye DiI and added to mSR-BI cells. Cells take up HDL via SR-BI 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. The uptake of lipid particles can be inhibited by the compound BLT-1 or when co-treated with an excess of unlabeled HDL. The mSR-BI cells utilized in the assay are a CHO cell line lacking expression of the LDL receptors and overexpresses the scavenger receptor, SR-BI.
Expected Outcome: Inhibitors of SR-BI and HDL uptake will have a reduction in fluorescence and activators will have an increase in fluorescence.
ldlA[mSR-BI] cells are a modified Chinese hamster ovary cell line that overexpress the SR-BI receptor. When maintaining cultures, it is important to fluid change cells every 2 days and avoid reaching 100% confluency. Periodically, cells are sorted using flow cytometry to maintain optimal levels of mSR-BI expression.
Plate 10,000 cells ldlA[mSR-BI], 30 ul per well in Ham's F12K/5% fetal bovine serum/Penicillin-Streptomycin-Glutamine using a Thermo Combi Multi-drop fluid dispenser. Use Aurora clear botttom, black walled, image quality 384 well plates. Incubate overnight at 37 degrees Celsius in a 5% CO2, humidified cell culture incubator.
1) Remove media with aspirator, rinse once with PBS (containing Mg & Calcium). Dispensing of PBS is done with Thermo Combi on slow setting to minimize disruption of cell monolayer.
2) Add 30 ul Ham's F12/0.5% Bovine Serum Albumin (fatty acid-free)/25 mM HEPES pH 7.4 + 10 ug protein/mL DiI-HDL with Thermo Combi fluid dispenser (slow setting)
3) Pin transfer 100 nl compounds, DMSO (neutral controls) and 1 uM BLT-1 (inhibitor control).
4) Incubate 3 hours @ 37 degrees Celsius in humidfied cell culture incubator
5) Remove media with aspirator, wash 2x with 1x PBS (containing Mg & Calcium). Dispensing of PBS is done with Thermo Combi on slow setting.
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=104) and positive control wells (PC; n=20) 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 (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.
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
Assay Cell Type: CHO
Assay Format: Cell-based
Assay Type: Functional
* Activity Concentration. ** Test Concentration.
Data Table (Concise)