Control Cell Fusion Counterscreen Assay Measured in Cell-Based System Using Plate Reader - 7013-02_Inhibitor_Dose_DryPowder_Activity
Assay Overview: The primary assay for this probe discovery project used a modified HeLa cell line that inducibly expresses the Envs of a primary HIV-1 strain (HIV-1JRFL) co-cultivated with receptor-bearing target cells (CEM21). The current specificity assay was built to exclude any cytotoxic or non-specific inhibitors that could appear as false positives in the cell-cell Env-mediated fusion more ..
BioActive Compound: 1
Keywords: cell fusion, luciferase, viral entry
Assay Overview: The primary assay for this probe discovery project used a modified HeLa cell line that inducibly expresses the Envs of a primary HIV-1 strain (HIV-1JRFL) co-cultivated with receptor-bearing target cells (CEM21). The current specificity assay was built to exclude any cytotoxic or non-specific inhibitors that could appear as false positives in the cell-cell Env-mediated fusion primary assay. For this purpose, we generated control HeLa-F-luc cells (referred to at CTL) that express the tetracycline activator (tTA) and carry the firefly luciferase (F-luc) gene under the control of a tTA-responsive promoter as effector cells. These cells are induced to express F-luc by withdrawal of doxycycline and are used to exclude any off-target inhibitors. The positive control used was doxycycline added to a final concentration of 40 ug/mL.
Expected Outcome: Env specific fusion inhibitors are expected to inhibit the cell-cell fusion system in the primary assay without affecting F-luc activity in the HeLa-F-luc (CTL) cells, whereas cytotoxic compounds are expected to decrease F-luc activity in both this assay and the primary assay. Therefore, only non-specific fusion inhibitors or cytotoxic compounds will lead to a decrease in luciferase signal in this counterscreen. Compounds that will be considered for probe development must have an IC50 at least ten times the IC50 in the primary assay.
Control (CTL) Fusion Assay Protocol
1.Grow CTL effector cells in DMEM passage medium with Doxycycline. Split 1:3 or 1:4 for passing. For HTS: 4 days prior to plating, thaw 3 vials (1.0 x 10;7 cells/ vial) into 4 triple flasks with 120 mL DMEM passage medium/flask. After 3 days, transfer to 3 Hyperflasks (HF) with ~550 mL RPMI induction media (~33 million cells per HF)
2.Day 0: Pass JRFL cells to DoxFree. Wash cells 2x with PBS & add 15 ml Accutase per triple to detach cells. Stop Accutase by adding RPMI induction media. Spin down cells & seed 9-10 x10;6 cells in 120 ml of RPMI induction medium in triple flask.
3.Day 1: Plating of CTL cells. Wash cells with PBS & add 15 ml Accutase for triple or 50 ml for HF to detach cells (@ 37 degrees C). Stop by adding RPMI induction medium. Spin down the cells at 1200 rpm for 5 min & re-suspend in phenol red-free RPMI induction medium (with Puromycin).
4.Count cells, adjust to 1.25 x10;5 cells/mL & seed 2,500 cells per well in 20 microl of RPMI (no Phenol Red) induction medium in Aurora white, opaque 384 well plates.
5.Day 2: Pinning & adding CEM21 cells Spin down & count CEM21 cells. Adjust to 5.0x105 cells/ml in RPMI fusion medium. Dispense 5,000 cells at 10 microl/well in RPMI induction media. Pin transfer compounds to the plate. Positive control is Maraviroc to a final concentration of 300 nM. Incubate the plate 37 degrees C overnight.
6.Day 3: SteadyGlo & Read. Add 10 microl of room temperature Steady-Glo substrate. Shake plate with Big Bear plate shaker. Incubate at room temperature for 10 minutes & read luminescence on a Perkin-Elmer EnVIsion with ultrasensitive luminescence settings for 0.5 seconds per well.
DMEM passage medium (1 L):
DMEM 882 mL
Heat Inactivated-FBS 100 mL
Pen-Strep-L-Glu 10 mL
*Doxycycline (1 mg/mL) mL
Puromycin (10 mg/mL)100 microL
Geniticin (50 mg/mL)4 mL
RPMI induction medium (DoxFree) (1 L):
RPMI (-Phenol red)890 mL
10 % Tet approved HI-FBS 100 mL
Pen-Strep-L-Glu 10 mL
Puromycin (10 mg/mL)100 microL
.Doxycycline is light sensitive, minimize light exposure
.All cells have to be in exponential growth phase. Hela-F-Luc cells are usually split 1:3 every 2-3 days, CEM#21 are split 1:10 every 3 days. Cells should not be allowed to overgrow in the flasks.
.CEM21 cells take 1-2 weeks after thawing to grow at an optimal rate
.Cells must be spun down after dissociation to remove StemPro Accutase from the medium.
.Cells can be allowed to fuse 16 to 24 hours.
.Effector (CTL) cells should be propagated for no more than 30 passages or 3 months whichever is sooner.
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 (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)
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)