Tissue tearor handheld homogenizer

Immunized mice were challenged one week after the last boost with 10⁵colony-forming units (CFU) of Listeria monocytogenes (Lm-GFP strain) (200
µL) by intraperitoneal injection. Negative controls included mice immunized with
sterile PBS and mice immunized with the nanofibers lacking a T-cell epitope. Mice
receiving a therapeutic TNF-neutralizing monoclonal antibody (500 µg, clone
MP6-XT22, Biolegend) 3 hours before Listeria monocytogenes challenge were
employed as the positive control. The spleen and liver were harvested 48h later. The
organs were placed in 5 mL sterile 0.05% Tween-20 in water, diced with scissors,
and homogenized with a Tissue Tearor hand held homogenizer (Biospec Products). The
homogenized sample (5 mL for the spleen or 6 mL for the liver) was serially diluted 3
times at 1:10 dilution in sterile 0.05% Tween-20 in water. 100 µL (spleen)
or 120 µL (liver) of undiluted, 10-, 100-, and 1000-fold diluted solutions were
plated on Brain-Heart Infusion Agar (BD Biosciences) in quadrant-divided Petri dishes. The
plates were allowed to air dry inside a sterile hood, then incubated upside down for up to
48 h in a 37°C incubator. The off-white Listeria colonies were
counted and the total CFU per organ were calculated using the formula: Total CFU= CFU
count × dilution factor × 50 (corrected for total organ homogenate
volume).

Spleens, livers, transplanted hearts and native hearts were isolated from each animal at 48 hours post Lm infection. Tissues were homogenized first with a surgical blade and then with a Tissue Tearor hand-held homogenizer (Biospec Products) in 0.05% Tween in water. Serial dilutions of the homogenates were plated on Brain Heart Infusion agar (BD) and incubated at 37°C for 24 hours before colonies were counted.