Multiplex elisa

Cytokines were measured by conventional ELISA (R&D Systems, BD, and elisakit.com) or multiplex ELISA (Quansys Biosciences). Both methods were performed as recommended by the manufacturer. If appropriate, cytokine data were normalized to total protein data (Pierce BCA Protein Assay Kit; Thermo Scientific) with the following formula: raw cytokine data (in pg/ml) / total protein concentration in the lysate (in mg/ml).

PBMC isolation and cytokine analysis were performed as described previously [66 (link)]. Briefly, NANPs alone, NANPs after complexation with Lipofectamine 2000 or generation 5 amine-terminated PAMAM dendrimers, and positive or negative controls were added to PBMC cultures, and the incubation continued overnight at 37 °C in an incubator with 5% CO₂. Complexation with Lipofectamine was done using the protocol described by us earlier [66 (link)]. For complexation with dendrimers, stocks of NANPs and dendrimers were incubated at room temperature for 30 min, then diluted in the complete cell culture medium (RPMI supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin). The final concentration of NANPs in the culture was 10 nM for all tested conditions (without a carrier, complexed with L2K, and complexed with dendrimers). After the incubation, the culture supernatants were collected and centrifuged at 18,000× g for 5 min. The supernatants were then analyzed by multiplex ELISA (Quansys Biosciences, Logan, UT, USA) to determine levels of individual cytokines.

The cytokines/chemokines release from whole blood cultures were measured as described previously [64 ]. Briefly, different dilutions of NSSL-MPS (0.304, 1.52, 7.62, 76.2 µg/mL MPS), positive and negative controls were incubated for 24 h (37 °C, 5% CO2 incubator, NuAire, Plymouth, MN, USA) in whole blood of at least 3 different healthy donors diluted 1:4 in complete culture media (RPMI with 10% heat inactivated FBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin). After the incubation, the cultured blood samples were collected and centrifuged at 18,000× g for 5 min. The supernatants were analyzed by multiplex ELISA (Quansys Biosciences, Logan, UT, USA) to determine levels of individual cytokines.

To determine the effect of gene editing on cytokine secretion, we used the Q-Plex Human Cytokine Array, 4-Plex, and a custom plate (Quansys Biosciences Multiplex ELISA, West Logan, UT, USA)^{70 (link)}. Concentrations of cytokines were determined by Quasys Q-View imaging and a software system.