HDL Uptake counterscreen Measured in Cell-Based System Using Plate Reader - 2085-07_Inhibitor_Dose_DryPowder_Activity
Assay Overview: In the primary assay, the cell line used to measure DiI-HDL uptake lacks the LDL receptor and overexpresses SR-B1. In this assay the parental CHO cell line that lacks the LDL receptor but does not overexpress SR-B1 will be used to determine if any uptake activators work via non-SR-B1 mechanisms. As in the primary assay, DiI-HDL will be used to measure uptake into cells. There should be little to no uptake and the only signal should be minimal background staining. ..more
Keywords: HDL, uptake
Assay Overview: In the primary assay, the cell line used to measure DiI-HDL uptake lacks the LDL receptor and overexpresses SR-B1. In this assay the parental CHO cell line that lacks the LDL receptor but does not overexpress SR-B1 will be used to determine if any uptake activators work via non-SR-B1 mechanisms. As in the primary assay, DiI-HDL will be used to measure uptake into cells. There should be little to no uptake and the only signal should be minimal background staining.
Expected Outcome: Some compounds might lead to an increase in DiI uptake because they alter endocytosis or some other cellular uptake mechanisms. A specific activator of SR-B1 uptake will have little to no activity in this assay. Only compounds with an IC50 >30 uM will be eligible as a probe.
ldlA7 cells are a modified Chinese hamster ovary cell line that lack the LDL receptor and were the parental line for the generation of the mSR-BI cell line used in the primary HTS. When maintaining cultures, it is important to fluid change cells every 2 days and avoid reaching 100% confluency.
Plate 10,000 cells ldlA7, 30 ul per well in Ham's F12K/5% fetal bovine serum/Penicillin-Streptomycin-Glutamine. 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/mL DiI-HDL with Thermo Combi fluid dispenser (slow setting)
3) Pin transfer 100 nl compounds, DMSO (neutral controls) and 1 uM BLT-1 (positive 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 Perkin Elmer 'Envision' plate reader with the following filters and mirrors: Bodipy TMR mirror #405, Excitation filter is Photometric 550 (#312) and emission filter is Cy3 595 (#229) using the bottom read setting.
PRESENCE OF CONTROLS: Neutral control wells (NC; n=104) 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' 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)