U bottom 96 well plate

We adapted and optimized an SBA assay based on formats described in the literature (22 (link), 37 (link)). Heat-inactivated serum samples were added to 96-well U-bottom plates (Fisher Scientific, Hampton, NH) and serially diluted 2-fold in PBS. S. flexneri 2a 2457T (500 CFU in 10 μl) and 25 μl of baby rabbit complement (BRC; Pel Biologicals, Rogers, AR) were added to the wells (for a total reaction volume of 110 μl and an initial serum dilution of 1:200), and plates were incubated for 1 h at 37°C in a shaker at 200 rpm. The numbers of viable CFU were determined by plating 10 μl of the reaction mixture on TSA and counting the colonies after overnight 37°C incubation. Negative-control wells containing only bacteria and BRC (no serum), as well as a positive-control serum with high bactericidal activity (mean SBA titer ± SD = 2,815 ± 237), were included in each assay. The percentage of killed organisms (per well) was determined by the equation [1 − (number of surviving bacteria/total number of bacteria)] × 100. SBA titers were determined as the reciprocal of the serum dilution that produced 50% bacterial killing using Reed-Muench regression analysis (38 ). Serum samples were tested and plated in duplicate (for a total of four independent CFU counts); these four data points per sample dilution were averaged for titer calculation.

To test the functionality of cTfh cells in supporting B cell differentiation in vitro, sorted memory B cells (2 × 10⁴) were cocultured with 2 × 10⁴ autologous CXCR3⁺ Tfh cells or 2 × 10⁴ autologous CXCR3⁻ Tfh cells in 96-well U-bottom plates (Thermo Fisher Scientific, Waltham, MA, USA) in RPMI1640 medium supplemented with 10% fetal bovine serum in the presence of endotoxin-reduced staphylococcal enterotoxin B (SEB) (1 µg/ml) (Toxin Technology, Sarasota, FL, USA). On day 7, the levels of IgM, IgG and IgA produced in the culture supernatants were determined with corresponding ELISA kits (Thermo Fisher Scientific, Waltham, MA, USA). Plasma cells (CD3⁻ CD19⁺ CD38⁺ CD138⁺) and HCV E2-specific B cells (CD3⁻ CD19⁺ HCV E2c Probe⁺) were analyzed by flow cytometry after 7 days of coculture, to assess the helper function of CXCR3⁺ and CXCR3⁻ Tfh cells with autologous memory B cells.

The hydrophobicity of A. butzleri in the presence or absence of bile salts was measured by a salting-out method as described by Misawa and Blaser (2000), with some modifications [61 (link)]. After overnight culture, the strain was transferred to a new culture in glass tubes without or with 0.2% bile salts and the tubes were incubated for 6 h at 37 °C with shaking at 250 rpm, under microaerobic conditions. Subsequently, the incubated strains were resuspended in 2 mM sodium phosphate (Carlo Erba, Val-de-Reuil, France) at an optical density at 620 nm (OD_620nm) of 1. Successive dilutions of ammonium sulfate (Labkem, Zelienople, PA, USA) at 4 M were carried out in 2 mM sodium phosphate in 96-well U-bottom plates (Thermo Scientific, Waltham, MA, USA). A volume of 25 μL of bacterial suspension was added to each well to a final volume of 50 μL. The plates were incubated at 25 °C (room temperature) overnight. After incubation, the minimum concentration of ammonium sulphate forming bacterial aggregation was determined by evaluation at naked eye, and the value was used for the hydrophobicity index. Hydrophobicity is inversely correlated with ammonium sulfate concentration causing bacterial aggregation. This test was performed at least three independent times.

To determine the cytokine secretion capacity of Tfh cells, PBMCs or sorted Tfh cells were seeded in 96-well U-bottom plates (Thermo Fisher Scientific, Waltham, MA, USA) and stimulated with 50 ng/mL PMA (Sigma-Aldrich, St. Louis, MO, USA) and 1 μM/mL ionomycin (Sigma-Aldrich, St. Louis, MO, USA) for 5 hours. GolgiStop (BD Biosciences, Franklin Lake, NJ, USA) was added 1 hour after stimulation. For flow cytometry assessment, cells were stained with surface antibodies and fixed using a Cytofix/Cytoperm fixation solution (Beckman Coulter, Brea, CA, USA) for intracellular cytokine staining according to the manufacturer’s recommendations.

Six-to-eight-week-old female CD1 mice (Harlan, Indianapolis, IN) were infected by intraperitoneal injection of 5 × 10⁸ CFU of S. aureus or S. epidermidis. Four hours postinfection, the animals were euthanized, and blood obtained after cardiac puncture was pooled from 4 mice into ice-cold sodium citrate at 0.35% (weight:vol) final concentration. Eukaryotic cells were lysed with 1% NP40 (Thermo Fisher Scientific, Waltham, MA), and bacteria were recovered by centrifugation (10 min) at 8 000 r · min⁻¹ at 4 °C. The bacterial pellet was sonicated in 2 mL ice-cold PBS, washed once in PBS, and transferred to a 96-well U-bottom plate (Thermo Fisher Scientific). First, non-specific binding to protein A was blocked with rabbit anti-protein A immune sera (1:100) for 30 min at 4 °C. Bacteria were then incubated with anti-LTA mAb or IgG1 (10 µg · mL⁻¹) for 1 h at 4 °C, washed in PBS, and incubated with Alexa Fluor 633 (Thermo Fisher Scientific)-conjugated goat anti-human IgG for 30 min at 4 °C (Jackson Immunoresearch Laboratory, West Grove, PA). Following one wash, live bacteria were stained for 15 min at room temperature with BODIPY FL Vancomycin and mAb binding was quantified as above.

The efficiency of the mKate knock-in was analysed on a CytoFLEX flow cytometer. The cells were washed twice with PBS 72 h after transfection or 96 h after electroporation and dissociated with TrypLE express enzyme (Thermo Fisher Scientific) or Accutase. The cell suspension was then transferred to a 96-well U-bottom plate (Thermo Fisher Scientific) and centrifuged at 300g for 5 min. The supernatant was aspirated, the pelleted cells were resuspended in 50 µl of 4% FBS in PBS and the cells were analysed on a CytoFLEX flow cytometer within 30 min following preparation.