Hand held tissue homogenizer

Six days or seven days after challenge, separated mice (n = 4/group) were sacrificed and their lung tissue was collected in 3 ml PBS containing 2% gentamycin. Collected lungs were homogenized for approximately 1 minute using a hand-held tissue homogenizer (Biospec Products, Bartlesville, OK, USA), and centrifuged to remove debris. The resulting supernatant was mixed with 10-fold diluted MDCK cell monolayers in 96-well tissue culture plates and incubated for 2 days at 37°C. The virus titer was calculated using the Reed-Muench formula and was expressed as log10 TCID₅₀ (median tissue culture infective dose) per milliliter.

Four days after challenge, mice (n = 2/group) were sacrified and their lung tissue was collected in 3 ml PBS containing 2% gentamycin. Collected lungs were homogenized for approximately 2 minutes using a hand-held tissue homogenizer (Biospec Products, Bartlesville, OK, USA) and centrifuged to remove debris. The supernatant was serially 10-fold diluted and transferred to MDCK cell monolayers in 96-well tissue culture plates and incubated for 2 days at 37°C. The virus titer was calculated using the Reed-Muench formula and was expressed as log10 TCID₅₀ per milliliter.