In vitro Targetability Bioassay: Measuring Intracellular cAMP in Living Cells With the Luminescent cAMP Probe

The experimental protocol and approaches to data processing are detailed in our earlier work (Paramonov et al., 2018). In brief, the cells with stable expression of GloSensor-22F cAMP probe (sensor cells) were seeded 1 day before the experiment into tissue culture-treated polystyrene 96-well plates with light-tight walls and translucent bottom (ViewPlate-96, PerkinElmer, Cat#6005181) as 60,000 cells per well in the 150 μl of cell type-specific medium, and incubated ON (+37°C in humidified atmosphere with 5% CO₂). The next day, before the assay, the old culture medium was removed and the wells were refilled with 45 μl of the freshly prepared inducing medium (IndMed), composed of 2% v/v of GloSensor reagent (Promega, #E1290; corresponds to the final working concentration of 0.612 mg/ml, with the original stock of 30,6 mg/ml in 10 mM HEPES, pH 7.5) and 200 μM of a non-specific phosphodiesterase inhibitor 3-Isobutyl-1-methylxanthine (IMBX; Sigma, #I5879) in the assay-specific medium. In case of MSN corona studies, DMEM/F-12 medium (50/50, v/v) with 10% (w/v) of iFBS was used for that purpose (aka Med_10%FBS, yielding IndMed_10%FBS); for other setups, a mix of the above medium and CO₂-independent medium (Gibco, #18045-054; 4v of DMEM/F12 per 5v of CO₂-independent medium), supplemented with 0.1% (w/v) of bovine serum albumin (BSA), was used (aka Med_0.1%BSA, yielding IndMed_0.1%BSA). After equilibration for 45 min at RT in the dark, the plate was inserted into a multiwell plate reader (EnSight, PerkinElmer, USA) and the light output—denoted as a baseline signal—was captured in a kinetic fashion, i.e., the selected wells on a plate were repeatedly captured in a desired sequence, for 15–20 min at RT. Next, the plate was removed from the reader and the wells were spiked with either 5 μl of freshly prepared solutions, having all the desired components at 10× of the final concentration, or respective controls. Final concentration of FSK in the assay equaled 10 μM, if not specified otherwise. FSK was not subjected to heat exposure or centrifugation (in the context of peptide/MSN thermal stability or peptide shedding assays, respectively), but its working solutions were prepared simultaneously with the actual study preps and kept for the same time at RT before being mixed with the actual preps to yield the final 10× co-mixes. After spiking, the plate was immediately re-inserted into the reader and the luminescence (now denoted as induced signal) was further captured in the same kinetic mode for the time required (typically, for 45–60 min). The described assay conditions (i.e., at RT, IndMed with 2% of GloSensor reagent and 200 μM of IBMX, stimulation with 10 μM of FSK) are referred to as “standard” throughout the text.

The registered luminescent reads were used for plotting of intracellular cAMP kinetic curves (luminescence vs. time), with the latter processed to baseline signal—subtracted area under the curve (AUC) values with the help of a custom-written script (available from the authors upon request). The derived AUC values were further normalized to the AUC of FSK, taken for 100% (if not specified otherwise), and the resulting %FSK-AUC indices were used for inferential statistics.