5 protocols
AccessionNameType
P-GSE43863-5
array scanning protocol
GeneChips were scanned using the Affymetrix 3000 7G Scanner.
P-GSE43863-4
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.
P-GSE43863-3
labelling protocol
The amplified cDNA was fragmented and labeled using the Encore Biotin Module
P-GSE43863-2
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.
P-GSE43863-1
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.