5 protocols
array scanning protocol
GeneChips were scanned using the Affymetrix 3000 7G Scanner.
hybridization protocol
Following fragmentation, cRNA were hybridized for 16 hr at 45C on GeneChip Mouse 430 2.0 Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.
labelling protocol
The amplified cDNA was fragmented and labeled using the Encore Biotin Module
nucleic acid extraction protocol
Extraction of total RNA was performed using the Qiagen RNA-mini kit according to the manufacturer's instructions. Genomic DNA was removed using DNAse treatment for some samples and qiagen RNA-mini plus columns for others. For RNA amplification, Ovation Pico WTA System (Nugen) for Affymetrix GeneChip® Arrays was used.
growth protocol
Congenically labeled (CD45.1) transgenic CD4 T cell splenocytes specific to the gp66-77 epitope of the lymphocytic choriomeningitis virus (LCMV) were obtained from naïve SMARTA TCR transgenic mice (Oxenius et al., 1998). For generation of SMARTA chimeric mice,naïve SMARTA CD4 T cell splenocytes were intravenously transferred into naïve C57BL/6 (CD45.2) mice. ~24 hours post-transfer, chimeric mice were infected i.p. with 2x10e5 PFU of LCMV Armstrong to establish an acute infection. For adoptive transfer of memory SMARTA cells, CD4+ splenocytes from chimeric mice (between 56 and 101 days post-LCMV infection) were enriched using a MACS CD4+ T cell Isolation Kit II (Miltenyi). Enriched CD4 T cells were then stained and sorted to isolate CD45.2- (CD45.1 SMARTA) Ly6chi CXCR5- and CXCR5hi Ly6clo populations to greater than 98% purity. 8x10e3 sorted memory SMARTA cells were adoptively transferred into naïve C57BL/6 mice (CD45.2) mice that were subsequently infected 24 hours post transfer with 2x105 PFU of LCMV Armstrong i.p.