GSM4007248: AB6206; Mus musculus; RNA-Seq
Mice were housed under specific-pathogen-free conditions at the Animal Breeding Center of the Weizmann Institute of Science, and were used at 6 to 8 weeks of age. All animals were handled according to the regulations formulated by the Institutional Animal Care and Use Committee. For the in vitro co-culture experiment, cells were isolated from spleens of C57BL/6 8 week old mice. DC derived from spleen tissues were enriched from the single cell suspension first by negative magnetic selection for CD19, and second by positive magnetic selection for CD11c. 2x106 DC were seeded in 6-well plate for 2h activation with 100ng/ml lipopolysaccharide (LPS; Sigma-Aldrich) and 2ug/ml OVA 323-339 (InvivoGen), at 37oC. In parallel, we isolated CD4+ T cells from spleens of OT-II mice. T cells were enriched by the CD4+ T cell Isolation Kit. For culture experiments CD11c+ DC and CD4+ T cells were plated on 96-well U-shape bottom, tissue culture plates. Co-cultured and mono-cultured cells were seeded in concentration of 1×106 cells/ml (1:1 ration in co-cultures), and harvested at different time points following cell plating (3h, 20h, 44h). For the in vivo infection experiment, N. brasiliensis (Nb) infective L3 larvae were prepared, washed in sterile PBS and killed by three freeze-thaw cycles. Mice were anesthetized and antigen was administered by intradermal injection into the ear pinna. PBS was injected into the ear pinna of control animals. Experiments were performed 48h following antigen injection.  To achieve single cell suspensions, auricular LN were digested in IMDM (Sigma-Aldrich) containing Liberase-TL (100μg/ml, Roche) and DNase-I (100μg/ml, Roche), and incubated with frequent agitation at 37oC for 20 min. Cells were washed with IMDM, filtered through a 100μm cell strainer, and centrifuged at 380g, 5min, 4oC. In both experiments, prior to sorting, cells were stained with DAPI for evaluation of live/dead cells. For the sorting of T-DC PIC samples were gated for TCRβ+CD11c+, for sorting of T cells samples were gated for TCRβ+, and for the isolation of DC, samples were gated for CD11c+, after exclusion of dead cells (DAPI-), erythrocytes (TER119-) and B cells (CD19-). 3' end mRNA libraries were prepared for sequencing using MARS-seq (Jaitin et al, Science 2014)