Primary Screening

Scientific Background¶

Compound cytotoxicity is an important parameter to measure when developing potential human therapeutics.

Jurkat clone E6.1 was screened against a diverse collection of 3,520 compounds. All compounds were tested once at a 1 μM final concentration.

A luciferase-based cell proliferation/viability assay endpoint kit was used as the readout. The kit measures the amount of ATP present in the microtiter plate well. If ATP is not present, the catalytic conversion of luciferin into oxyluciferin is not possible, and no luminescence results. Since metabolically active cells produce ATP, the absence of ATP correlates with the presence of non-viable cells.

This campaign used doxorubicin, an antibiotic used as an anti-cancer drug, as the positive control. The assay was conducted in 384-well format.

To select a practical number of compounds that exhibited high activity in the primary screen for follow-up assays, a cutoff value of the primary %Inhibition was applied. The cutoff value was calculated as the sum of the average percent inhibition of all compounds tested and three times their standard deviation (= HITS).

Primary Screening Analysis with ASSAY

Prerequisites¶

An existing run template, created using Design Mode, following the tutorial in Primary Screening Tutorial.

Files to use:

Run Creation¶

Click the New Run button to create a new run.
Select Execution Mode as the mode.
Select the appropriate Run Template (the one saved in the Publisher Step of Design Mode).
Enter a Run Name.
Go to the File Reading step.

File Reading¶

The reader named Analyst GT (TAB) is already fixed.
Upload the file 01-Primary_Screening_10p384raw_data.
Go to the Raw Data File step.

Raw Data File¶

Number of measurements per plate is already mapped: 1.
The Readout Alias is fixed to READOUT1 and cannot be modified.
Go to the Plate Mapping step.

Plate Mapping¶

The Control Layout named Primary_Screening is already applied to all plates.

Go to the Well Validation step.

Well Validation¶

The visualization template selected in Design Mode is already applied. You can change this template.

For QC Validation, select aberrant wells in the visualization and click on the Well Validation step: Invalidation button button. Multiple selection is allowed by holding the Ctrl key.

Example

Plate_ZSCORE_READOUT1 < -2 or Plate_ZSCORE_READOUT1 > 2.

Go to the Sample Mapping step.

Sample Mapping¶

Click on the Edit Sample Information button.
Select filling mode File and tick Sample and Dose for content.
Choose file 01_Sample_Mapping_Primary_Screening.
Click on the Apply button.

Samples and doses are filled in.

Click on the Continue button. Samples and doses are mapped for all plates.
Click on the Sample Analysis Settings button.

Sample Analysis Settings¶

Groups are automatically created (3,520 groups).
Basic Statistics analysis is already applied.
Go to Sample Analysis.

Sample Analysis¶

The template specified in Design mode is applied.

For Hits Selection, select points (“hits”) in visualization and add them to a list (click on the Add Hits button). This selection can be exported in CSV format (export button).
Lock your Analysis (Lock Run button).

Publish Data¶

Depends on the Publisher defined in the run template.