96 well flat bottom microplate

The phagocytic and bactericidal activities of splenic macrophages were determined as described previously [88 (link)]. Splenic macrophages were prepared by adhering spleen cells twice in RPMI 1640 medium (Thermo Fisher Scientific) supplemented with 10% FBS in a petri dish for 1 h at 37 °C under 5% CO₂ conditions. Splenic macrophages, resuspended in antibiotic-free RPMI 1640 medium supplemented with 10% FBS at a concentration of 10⁶ cells/mL, were infected with L. monocytogenes at MOI of 10, followed by incubation at 37 °C for 30 min. Subsequently, macrophages were washed three times with RPMI 1640 medium supplemented with 10% FBS and 50 µg/mL gentamicin and were transferred into a 96-well flat-bottom microplate (Nunc, Roskilde, Denmark) at a density of 10⁶ cells in 100 µL per well. The infected cells were lysed by treatment with RPMI 1640 medium containing 1% (wt/vol) CHAPS at 0, 2, 4, and 6 h. Lysates from three wells were pooled, and the number of viable intracellular bacteria in each sample was determined by culturing on a tryptic soy agar plate.

Overnight cultures were diluted 1:200 in either TSB or CS2 and 200 μL was transferred to a 96-well flat-bottom microplate (Thermo Scientific), which was then incubated in a multimode plate reader (2300 Enspire^TM, PerkinElmer^®) with shaking at 37 °C. OD₆₀₀ was measured every 30 min.

Blood samples (8 mL) were collected from seven healthy donors. All blood samples were commercially obtained from the Regional Blood Centre in Warsaw. The isolation of peripheral blood mononuclear cells (PBMCs) was performed via density gradient centrifugation on a Histopaque-1077 (Sigma-Aldrich, St. Louis, MO, USA). PBMCs were collected at the interface between the plasma and the Histopaque and washed twice with phosphate buffered saline (PBS, Sigma-Aldrich, St. Louis, MO, USA). The separated PBMCs were resuspended in RPMI 1640 medium (Gibco, Gaithersburg, MD, USA) containing antibiotic-antimycotic solution (1.5% penicillin-streptomycin-amphotericin, Invitrogen, Waltham, MA, USA) and 10% human serum (Gibco, Gaithersburg, MD, USA) and counted. The PBMCs (2 × 105 cells/well) were cultured in 96-well flat-bottom microplates (Nunc, Paris, France), as described in detail in our previous paper [43 (link)]. Briefly, the cells were stimulated with anti-CD3 Ab (coated on plate wells, 0.75 μg/mL, BD Pharmingen, Franklin Lakes, NJ, USA) or Dynabeads™ Human T-Activator CD3/CD28 (2 μL per well, ratio 2:5, Gibco, Gaithersburg, MA, USA) and incubated with L. edodes mycelium extracts at a concentration of 100 µg/mL. The PBMCs were cultured for 24 h at 37 °C and 5% CO₂ in a humidified incubator.

The following plasma proteins were measured with MagPlex Luminex assays (Bio-techne) using a Luminex™ FLEXMAP 3D™ (Luminex): Angiogenin, Angiopoietin-1, Angiopoietin-2, BDNF, CD30, CD40 Ligand, CTACK, Flt3 Ligand, Eotaxin, HGF, IL-1ra, IL2Rα, IL-6, IL-16, IL-17A, IL-18, MCP-1, MIP-3α, MIF, MIG, MIP-3β, MMP-1, MMP-8, MMP-12, MMP-13, PDGF-AA, PF4, RAGE, RANTES, SCGF, S100B, S100A9, TNF-α. Adiponectin, BDNF, calprotectin, CRP, M-CSF, MPO, PDGF-BB, TfR, VEGF were measured with Duoset ELISAs (Bio-techne) according to the manufacturer’s instructions in 96 well flat bottom microplates (Nunc). Plasma samples were run in duplicates and in all instances, paired pre and post samples were assayed on the same plate, with 20 paired samples run on the first batch and 10 paired samples run on the second batch.