Pha m

To stimulate PBMCs with fiber NANPs for the
assessment of cytokine
induction, cells were brought up to 1.25 × 10⁶ cells/mL
and seeded in 96-well U-bottomed plates with 200 μL per well.
RNA/DNA fibers at 1 μM stock solution were complexed to Lipofectamine
2000 (L2K) at a 5:1 v/v ratio. After a 30 min incubation at room temperature,
OptiMEM was added, bringing the final concentration of fibers to 50
nM. Afterward, 40 μL of the prepared controls and NANPs were
added to PBMC for a final stimulation concentration of 10 nM. As the
positive controls, LPS (final 20 ng/mL, Invitrogen), ODN2216 (final
5 μg/mL), and PHA-M (final 10 μg/mL, Sigma) were added
to PBMCs. As a negative control, blank media was added to PBMCs. All
treatments were added to wells in technical duplicates for each donor.
After 20 h of incubation at 37 °C, the plate was spun down at
700g for 10 min. 170 μL of supernatant was
collected from each well and transferred into a new 96-well plate
for analysis of cytokines by multiplexed ELISA (Q-Plex Mouse Cytokine
– Inflammation (14-plex) and Q-Plex Human Cytokine –
HS Screen (15-plex) from Quansys Biosciences) according to the manufacturer’s
instructions. Supernatants from the three positive controls were pooled
1:1:1. Plates were imaged on a Quansys Biosciences Q-View Imager Pro.^{63 (link)}

Splenic CD4⁺ T cells were isolated using magnetic‐activated cell sorting (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's protocol. Briefly, positive selection of CD4⁺ T cells from 8‐week‐old C57BL/6 mouse spleens was performed according to the manufacturer's protocol using CD4 Microbeads (Miltenyi Biotec). CD4⁺ T cells (2 × 10⁵ cells/well), stimulated with phytohemagglutinin‐M (PHA‐M) (5 μg/ml, Sigma‐Aldrich, St. Louis, MO, USA), were co‐cultured with allogeneic BMDCs (2 × 10⁴ cells/well) in complete medium. For T cell proliferation assay, BMDCs were stimulated with Necrotic‐S or transfected with miRNA precursors or inhibitors. T cell proliferation was determined by thymidine incorporation on days 2, 5 and 7. To determine cell proliferation, cells were pulsed with [³H] thymidine during the last 18 hrs of culture and incorporated radioactivity was quantified using a liquid scintillation counter (Beckman, Fullerton, CA, USA).