Anti lcmv np antibody vl 4

Mice were infected intravenously (i.v.) with LCMV-Armstrong (2 × 10⁶ PFU/mouse), and their serum and tissues were collected at day 8 for analysis. Viral titer in the serum from infected mice was assessed using a modified focus-forming assay^{56 (link)}. Diluted serum (1:100) was incubated with Vero cells (2.5 × 10⁴ cells/well) on a 24-well plate at 37 °C for 4 hours. Each well was subsequently overlaid with 0.5% methylcellulose and incubated at 37 °C for 48 hours. Cells were subsequently fixed with 2% formalin/formaldehyde for 30 minutes and then with 0.5% Triton-X for 20 minutes. Fixed cells were stained with anti-LCMV NP antibody (VL-4; Bio X Cell, 5 μg/ml) for 1 hour, followed by staining with anti-rat IgG HRP antibody (Jackson Immunoresearch, dilution 1:1,350) for 1 hour. LCMV foci were visualized using an ImmPACT DAB Peroxidase (HRP) Substrate kit (Vector Labs).

Vero cells (African green monkey kidney cells originally acquired from the American Type Culture Collection ∼40 years ago) were propagated in Eagles MEM (Gibco) supplemented with 7% FBS, 100 g/mL penicillin/streptomycin (Gibco), 2 mM L-glutamine (Gibco). BHK-21 cells were grown 10% FBS, 100 g/mL penicillin/streptomycin, 2 mM L-glutamine, 7% tryptose phosphate broth solution (Sigma), and 0.56% glucose (wt/vol). The following anti-mouse antibodies were purchased from BD Biosciences: PE CD4 (RM4.5), PE IFNγ, PerCP-Cy5.5 CD3 (145-2c11), PE Ly6G (1A8), and PerCP-Cy5.5 NK1.1 (145-2c11). The following anti-mouse antibodies were purchased from eBioscience: FITC TNFα (MP6-X722), PE SiglecH (440C), PerCP-Cy5.5 CD11c (N418), PerCP-Cy5.5 Ly6c (HK1.4), e450 and APC CD8a (53–6.7), e450 B220 (RA3-6B2), e450 and PE-Cy7 CD90.2 (53–2.1), e450 and PE-Cy7 CD19 (1D3), PE-Cy7 CD3 (145-2c11), PE-Cy7 CD21/CD35, PE-Cy7 CD11b (M1/70), APC and APC-Cy7 CD45 (30F11), APC CD31 (390), and APC PDCA-1 (927). The following anti-mouse antibodies were purchased from BioLegend: PE gp38 (8.1.1), PE-Cy7 CD8a (53–6.7), and APC F4/80 (BM8). Anti-LCMV NP antibody (VL-4) was purchased from BioXcell and conjugated to Alexa Fluor 488 using antibody labeling kit from Life Technologies.