M1190 0000

Isolated cells from 3 individual donors were stimulated with a pan-bacterial cocktail comprised of lipopolysaccharide from Escherichia coli O26:B6 (LPS) (Sigma, L2654) at 1 µg/mL and heat-killed Staphylococcus aureus (HKSA) (InvivoGen, tlrl-hksa) at 10⁸ cells/mL. Commercial stocks were reconstituted at 100 × in UltraPure™ DNase/RNase-Free Distilled Water (Invitrogen, 10977015) and stored as individual use aliquots at − 20 °C. Unstimulated cells received 2 µL water as vehicle. Cells were incubated for 24 h in RPMI containing 1% human serum plus the relevant stimulus at 37 °C with gentle agitation at 80 rpm on an orbital shaker (Eppendorf, M1190-0000) housed inside a standard tissue culture incubator (5% CO₂, humidified). At endpoint, cells were collected by centrifugation at 400 g for 5 min at room temperature and supernatants were collected, clarified at > 16000 g for 5 min, and stored at − 20 °C.

Following cryorecovery, cells were resuspended in RPMI containing 1% human serum, counted, and plated at 2 × 10⁵ cells per well in 200 μL in ultra‐low attachment U‐bottom 96‐well plates (Corning, 7007). Cells were stimulated overnight with a pan‐bacterial cocktail comprised of lipopolysaccharide from Escherichia coli O26:B6 (LPS) (Sigma, L2654) at 1 μg/mL and heat‐killed Staphylococcus aureus (HKSA) (InvivoGen, tlrl‐hksa) at 10⁸ cells/mL, or with a pan‐viral cocktail comprised of high molecular weight (HMW) poly(I:C) (InvivoGen, tlrl‐pic), Lyovec encapsulated HMW poly(I:C) (InvivoGen, tlrl‐piclv), single‐stranded Poly(U) naked RNA (InvivoGen, tlrl‐sspu), and Lyovec encapsulated ssPoly(U) (InvivoGen, tlrl‐lpu), each at 1 μg/mL final concentration. Concentrated cocktails were added to stimulated cells at 1:100 dilution. Unstimulated cells received an equivalent amount of water as vehicle. Cells were incubated for 24 h at 37°C with gentle agitation at 80 rpm on an orbital shaker (Eppendorf, M1190‐0000) housed inside a standard tissue culture incubator (5% CO₂, humidified). Following stimulation, cells were centrifuged at 400g for 5 min at room temperature and supernatants were collected, clarified at >16,000g for 5 min, and stored at −20°C for analysis.