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For quantification purposes, a calibration curve of horseradish peroxidase (Calbiochem) ranging from 100 mU/ml to 3.13 mU/ml was run in parallel with the samples in triplicate with every experiment.
Positive and negative controls were set up with every experiment.
Seven control DNAs (supplementary table 2) with different genotypes were included with every experiment.
The water-only sham digest controls and no-template PCR controls used with every experiment were consistently negative confirming that we were able to control contamination originating from the DNA extraction and PCR set ups.
To confirm that these negative results were not due to lack of drug efficacy or some systematic problem, positive controls (P2X2 and GCaMP3.0 or EPAC expressed in the same cell type) were performed concurrently with every experiment (data not shown).
To control for inter-experimental variation in CD3 zeta-chain mean fluorescence, cells from a single donation, from a healthy donor, were cryopreserved in aliquots and one aliquot was thawed with every experiment to establish a normalization factor.
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But, as with every experiments from TheFamily, there are a lot of requirements and work ahead.
Then, we gathered the decision boundary lines of each experiment for determining the time of failure, and we verified every experiment with the most conservative decision boundary line.
N2 control was included in every experiment with nematode mutants.
The system was calibrated before every experiment with a test grating (12.5 μm).
A parallel reaction was set up for every experiment with the inhibitor of respective complexes to assure the specificity of the reaction.
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