Minimum essential medium (mem)

Nasal swabs were collected from each buffalo at d0, d2, d4, d7, d10, d15, d30, and d63 pc, placed in transport fluid Minimum Essential Medium (Euroclone, Milan, Italy), and used for BuHV-1 isolation and titration assays. Serial dilutions ranging from 10^–1–10^–9 of supernatants from each nasal swabbing were inoculated in a volume of 0.1 mL into three wells of a 24-well plastic plate containing monolayers of MDBK cell cultures grown in MEM. The cells were provided by Biobanking of Veterinary Resources (BVR), Brescia, Italy, and identified with the code BS CL63. After 60-min incubation at 37 °C in a 5% CO₂ atmosphere, 1 mL of MEM enriched with 2% FCS (BioWhittaker Inc., Walkersville, MD, USA) was added to each well. The positive control was prepared from MDBK cell cultures infected with the wt 799,787 MT strain of BuHV-1. MDBK cell cultures free of BuHV-1 were used as a negative control.
The plates were incubated for 7 days at 37 °C in a 5% CO₂ atmosphere and observed daily for the appearance of cytopathic effect (CPE). BuHV-1 titre was determined as previously described [17 (link)] and expressed as TCID₅₀/mL. The BuHV-1 recovered from each positive sample was identified by PCR [1 (link)].

B16/F10 melanoma (CRL-6475) and Lewis lung carcinoma (LLC-1, CRL-1642) cell lines were purchased from the American Tissue Culture Collection (Manassas,VA) and confirmed to be mycoplasma free. The cells were maintained in Minimum Essential Medium (Euroclone) supplemented with 10% (v/v) foetal bovine serum, 1% (v/v) penicillin–streptomycin solution, 2 mM L-glutamine, 100 μM non-essential amino acids, 1 mM sodium pyruvate and 10 mM HEPES (all from Life Technologies, Paisley, UK) in a humidified 5% CO₂ atmosphere at 37 °C. C57BL/6 WT, C57BL/6.C1qa^−/−, C57BL/6.C3^−/− or C57BL/6.C5^−/− were injected intramuscularly into the left flank of the mice with viable B16/F10 cells (2 × 10⁶) and tumour development was monitored daily. Both male and female mice (8–12 weeks of age) were used. In each experiment the mice were sex- and age-matched. Tumour size was measured every other day for the first week and then daily with calliper by determining two orthogonal axis, and the volume was calculated using the following formula: (π/6)·α²·β, with α the shorter and β the longer axis65 (link). Measurements were performed by a blinded researcher. According to our protocol (D.L.116/92), mice were culled before they developed any signs of distress. When the animals were killed, tumours, spleens and lungs were collected for analyses.

Nasal swabs were collected from each buffalo at d0, d2, d4, d7, d10, d15, d30, and d63 pc, placed in transport fluid Minimum Essential Medium (Euroclone, Milan, Italy), and used for BuHV-1 isolation and titration assays. Serial dilutions ranging from 10^–1–10^–9 of supernatants from each nasal swabbing were inoculated in a volume of 0.1 mL into three wells of a 24-well plastic plate containing monolayers of MDBK cell cultures grown in MEM. The cells were provided by Biobanking of Veterinary Resources (BVR), Brescia, Italy, and identified with the code BS CL63. After 60-min incubation at 37 °C in a 5% CO₂ atmosphere, 1 mL of MEM enriched with 2% FCS (BioWhittaker Inc., Walkersville, MD, USA) was added to each well. The positive control was prepared from MDBK cell cultures infected with the wt 799,787 MT strain of BuHV-1. MDBK cell cultures free of BuHV-1 were used as a negative control.
The plates were incubated for 7 days at 37 °C in a 5% CO₂ atmosphere and observed daily for the appearance of cytopathic effect (CPE). BuHV-1 titre was determined as previously described [17 (link)] and expressed as TCID₅₀/mL. The BuHV-1 recovered from each positive sample was identified by PCR [1 (link)].