HTS using DiI-HDL to assay lipid transfer in ldlA[SR-BI] cells Measured in Cell-Based System Using Plate Reader - 2085-01_Activator_SinglePoint_HTS_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: 306
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. Inhibtors 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) and positive control wells (PC) were included on every plate.
EXPECTED OUTCOME: Active compounds result in increasing readout signal.
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: The plate pattern correction algorithm 'Runwise Pattern (Multiplicative)' in Genedata (v7.0.3) was applied to the normalized plate data.
This was set as equal to the mean of the normalized and corrected sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at 60.
PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 100.
Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T
Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T
Samples passing AT only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T
tSamples passing PAR_T only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)