Normal mouse serum

A polysaccharide-specific Sandwich ELISA was employed to screen the anti C-Ps mAb against several PnPs serotypes as described previously [31 (link)]. In this ELISA assay, the plates were coated with a CRM capture mAb (1 μg/mL). At 2–8 °C, the coated plates were blocked and incubated overnight with serially diluted target PnPs serotypes. After the plate wash, the anti C-Ps mAb (1 μg/mL) was applied to the plates and they were incubated for one hour. The plates were washed, then incubated for one hour with 1:6000 dilution of donkey anti-human antibody conjugated with alkaline phosphatase (Jackson ImmunoResearch, West Grove, PA, USA) in a solution of assay diluent spiked with normal mouse serum (Jackson ImmunoResearch). The plates were washed, 4-Methylumbelliferyl-phosphate (4-MUP) (Virolabs, Chantilly, VA, USA) was applied, and they were incubated for 45 min. The fluorescence signal was recorded on a SpectraMax M2E spectrophotometer at 360 nm (excitation)/450 nm (emission). Data were collected using SoftMax Pro (Molecular Devices, San Jose, CA, USA).

Tumors were harvested at an average tumor size of 150-200 mm³ and snap frozen. Tissues were cryosectioned at 8 µm and immunohistochemical (IHC) experiments were conducted as follows: slides were fixed in cold acetone or 4% formalin, blocked in 5% normal mouse serum (Jackson Immunoresearch) and incubated with primary antibodies for 1.5 h at room temperature. Primary antibodies were anti-CD3 (Dako, A0452, #280), anti-CD4 (Affymetrix, #14-0042) and anti-CD8a (Affymetrix, #14-0081). Sections were dried and stained with haematoxylin/eosin in a Leica ST4040 automatic stainer.
Assessment of T cell infiltration was performed in a blinded fashion and scored as 0 (negative), 1 (<150 cells per mm²), 1-2 (150-300 cells per mm²), 2 (300-500 cells per mm², 2-3 (500-800 cells per mm²), and 3 (>800 cells per mm²⁾. N=3 per syngeneic tumor model.

For infections with S.Tm, the bacteria were pelleted and resuspended in PBS followed by measuring the OD₆₀₀ of the suspension. Bacteria for the infection were opsonized by taking 45 μL of bacterial culture, mixing with 20 μL of normal mouse serum (Jackson ImmunoResearch) and 135 μL of DMEM with 10% normal FBS (Gibco) for 20 minutes. This was followed by adding an additional 600 μl of DMEM with 10% normal FBS, and then adding the desired bacteria to the cells at an MOI of 10. The plate was spun at 100 × g for 5 minutes then incubation at 37°C for 30 minutes. The cells were washed three times with PBS, fixed with PFA 4% for 1 hour at room temperature. The cells were washed 3 times in PBS and mounted with Prolong mounting medium with DAPI (Molecular probes).
For infections with Mtb, the bacteria were pelleted and washed in PBS-tween80 0.05%. The OD600 was measured, and the bacteria were added on the cells at an MOI of 10. The plate was then spun at 200 × g for 5 minutes and incubated for 1 hour. The cells were washed three times with PBS and incubated with fresh complete medium. At the designated time post-infection, the cells were then washed three times with PBS, fixed with PFA 4% for 1 hour at room temperature. Finally, the cells were washed 3 times in PBS and mounted with Prolong mounting medium with DAPI.