HDL Counter Assay: Tf Endo Assay Measured in Cell-Based System Using Plate Reader - 2085-04_Inhibitor_Dose_DryPowder_Activity
This assay is used to determine the selectivity of SR-BI inhibitors versus other receptors. It measures the endocytosis of an independent ligand, Transferrin, which is not taken up via the SR-BI receptor. Transferrin binds the transferrin receptor. The occupied receptor is endocytosed, moves to the endosomal compartment and is cycled back to the cell membrane to retrieve more tranferrin. Cells more ..
Keywords: transferrin, endocytosis
This assay is used to determine the selectivity of SR-BI inhibitors versus other receptors. It measures the endocytosis of an independent ligand, Transferrin, which is not taken up via the SR-BI receptor. Transferrin binds the transferrin receptor. The occupied receptor is endocytosed, moves to the endosomal compartment and is cycled back to the cell membrane to retrieve more tranferrin. Cells are pre-treated with compound for 3 hours and stained with the Alexa-594 Transferrin reagent for 30 minutes. Analysis was performed using the translocation module within Metaexpress high content imaging software (Molecular Devices). Briefly, signal was measured adjacent to the nucleus within the cytoplasm where endosomes are located. The percent classified positive values were normalized and analyzed in Genedata Assay Analyzer & Condeseo.
Expected Outcome: It is known that uptake of HDL is independent of endocytosis and transferrin uptake is an endocytosis-mediated process, compounds that inhibit endocytosis (i.e. transferrin uptake) will not be of interest. Compounds that do not block uptake of transferrin will be considered for further studies.
Transferrin Endocytosis Protocol
Transferrin preparation: 5 mg dissolved in 1 mL water. Dilute 5 mg/ml stock 1:10 to become 5 ug/ul stock, add 1.8 ul per well for 30 ul volume (need ~750 uL per 384 well plate). Make sure the quick spin transferrin in microcentrifuge to remove any aggregates.
Plate 4000-5000 mSR-BI cells in 30 microl per well in Ham's F12K/5% FCS/Pen-Strep. Use black, clear bottom, Aurora image quality, tissue culture treated 384 well plates (PN 32441).
Wash with 2 X 40 microl DMEM/BSA/(no phenol red) to remove any serum (which can contain significant amounts of transferrin that can interfere with the assay). Any residual buffer needs to be removed.
Dispense 30 microl per well of DMEM/1%BSA/no phenol red
Add compounds by pinning 100 nL per well. Incubate 60' @ 37 degrees C
Dispense 20 microl fluorescent Transferrin-Alexa-594 (Invitrogen #T-13343) to a final concentration of 30 microg/ml. Incubate 30' @ 37 degrees C
Place plate on ice to stop reaction.Wash wells with 45 microl ice cold PBS/1mM MgCl2/0.1mM CaCl2
Fix for 45' on ice with ice cold 4% paraformaldehyde in PBS/1mM MgCl2/0.1mM CaCl2 in the presence of DAPI DNA dye (1:1000 from a 10mg/ml stock)
Plates were imaged on a Molecular Devices IXM microscope at 20x magnification for DAPI nuclear stain and Texas Red/Alexa fluor 594 fluorescence for the transferrin. Two sites were imaged per well. The Metaexpress software analysis tool for translocation was used to determine the location of intracellular Alexa fluor-594 labeled transferrin relative to the nucleus. Cells were analyzed for meeting a certain fluorescence signal threshold above background levels of fluorescence in the perinuclear cytoplasmic region surrounding the DAPI stained nuclei. Cells meeting or exceeding the fluorescence threshold were deemed positive. The number of cells per site was calculated using the DAPI stain. The percent of cells per site was calculated as the "percent classified positive" measurement.
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)