AID	Name	Type	Comment
651609	Broad Institute MLPCN HIV Entry Inhibitor Probe Project	summary	Summary assay

Keywords: CEM, lymphoblast cell line, cytotoxicity, luciferase, CellTiter-Glo

Assay Overview:
In the fusion assays, a reduction in luciferase signal could be a direct result of cell cytotoxicity and have nothing to do with inhibition of viral fusion. To discard such false positives, the cytotoxicity of each positive hit towards CEM cells will be evaluated using the CellTiter-Glo reagent. Cells will be treated with compounds at multiple doses for 24 hours. Cell viability will be measured using CellTiterGlo (Promega) which measures cellular ATP levels as a surrogate marker of cell viability/proliferation.

Expected Outcome: A dose-dependent decrease in luciferase signal will suggest that a compound is cytotoxic and will not be a viable probe candidate.

1.Grow CEM21 cells in RPMI induction medium. Split 1:3 or 1:4 for passing.
2.Day 1: Collect CEM21 cells in conical tubes. Spin down the cells at ~1200 rpm for 5 minutes & re-suspend in phenol red free RPMI induction medium (with Puromycin).
3.Count cells, adjust to 3x10;5 cells/mL & seed 3,000 cells per well in 30 uL of RPMI (no Phenol Red) induction medium in Corning 8867BC white 384 well plates.
4.Day 2: Pin transfer 100 nL of compounds to the plate. Incubate the plate 37 degrees C overnight.
5.Day 3: Add 20 uL of room temperature CellTiter-Glo (Promega). Wait 10 minutes & read luminescence with standard luminescence settings on a Perkin-Elmer EnVision plate reader.


Media Recipes

RPMI induction medium (500 mL):
RPMI (-Phenol red) 443 mL
Tet approved HI-FBS 50 mL
Pen-Strep-L-Glu 5 mL
Puromycin (10 mg/mL)50 uL


Notes
.All cells have to be in exponential growth phase. CEM21 cells are split 1:10 every 3 days. Cells should not be allowed to overgrow in the flasks.
.All incubations are done at 37 degrees C in the presence of 5% CO2.
.Cells should be propagated for no more than 30 passages or 3 months whichever is sooner.

PRESENCE OF CONTROLS: Neutral control wells (NC; n=32) were included on every plate.

EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
The compounds were assayed in multiple independent instances using an identical protocol; each instance is called a 'test'. For each test, the following analysis was applied:

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).

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls' method in Genedata Assay Analyzer (v10.0.2):
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.10.0.2) 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)

PUBCHEM_ACTIVITY_OUTCOME:
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.

PUBCHEM_ACTIVITY_SCORE:
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.

DATA AGGREGATION:
Once the data for each test was processed, the test number was appended to all column headers in that test's data set. The individual test results were then aggregated as follows:

1. The final PUBCHEM_ACTIVITY_OUTCOME was set to:
a. '2' (active) when all test outcomes were '2' (active), or
b. '1' (inactive) when all test outcomes were '1' (inactive), or
c. '3' (inconclusive) when the test outcomes were mixed.

2. The final ACTIVE_CONCENTRATION (AC) was set as follows:
a. If the final PUBCHEM_ACTIVITY_OUTCOME = 2, AC was set as the mean of the constituent test active concentrations;
b. If the final PUBCHEM_ACTIVITY_OUTCOME = 1 or 3, AC was left empty.

3. The final PUBCHEM_ACTIVITY_SCORE was calculated based on the aggregated ACTIVE_CONCENTRATION, using the same logic described above for individual test scores.

Note:
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.

Grant Number: 1 R03 DA034601-01