In order to obtain single-cell suspensions, lymph nodes (pLNs, PPs, and MLNs) were digested with 0.14 Wünsch U/ml Liberase C1 or Liberase TL (Roche Applied Science).
We prepared a single-cell suspension of lymph node cells (LNC) in PBS for each mouse by grinding the lymph nodes with the flat end of a 5-cc syringe.
This preliminary study showed, after immediate sampling on filter paper, that amplicons were obtained for both nasal swabs and blood samples but not for the lymph node suspension.
The draining lymph nodes (inguinal, para-aortic, and popliteal) of sub-groups of these rats were harvested on day 8, 12 or 24 after injection, and a single cell suspension of lymph node cells (LNCs) was prepared [ 32].
Each animal was sampled by using a lymph node suspension, a nasal swab, and a blood sample to smear filter papers, further tested by RT-PCR designed to amplify the F gene directly from filter paper.
Mice were sacrificed and single cell suspensions of lymph node and spleen were prepared.
The single cell lymph node suspensions were prepared immediately after excision of nodes from each mouse and kept in FACS Buffer with saturating amount of thymidine (20 mg/ml) on ice to block further incorporation of BrdU during excision of other samples.
Mixed cell suspensions of lymph node and spleen were plated in 24-well plates in complete IMDM supplemented with hIL2 (10 U/ml) and stimulated with irradiated (1600 rad) B16OVA cells (5*104 cells/well) that had been treated overnight with r-IFN-γ (50 U/ml) to up-regulate MHC class I presentation.
Single-cell suspensions from lymph node preparations cultured at 5×10 cells per mL.
Then, the lymph node suspensions were lysed with FACSTM Lysing Solution BD Biosciencess) for 10 min at room temperature in the dark.
In brief, CD3+ T-cells were positively enriched from single-cell lymph node suspensions using antibody (anti-CD3 -conjugated anti-CD3 -conjugated
