strain: B5 TCR Tg mice (BALB/cJ) subset group: Early Effector (E, CD62Lhi CD27hi) infection: day 8 post P. chabaudi-infection
Treatment protocol
Splenic CD4 T cells from naive or infected B5 TCR Tg mice ( 5- to 12-wks-old) were enriched by EasySep biotin selection kit (Stemcell Technologies, Vancouver, BC, Canada) with a mixture of biotinylated anti-CD8a, B220 (RA3-6B2), CD11b (MI/70), CD11c (N418), F4/80 (BM8), and Ter119 (eBioscience). The negative fraction was then stained with anti-CD4-FITC, CD44-allophycocyanin-Cy7, CD127-PE, anti CD62L PE-TxRd and CD27 APC for the subsets. Cells were sorted on a FACS Aria with FACDiva software (BDbiosciences, San Diego, CA). Effector (CD44int-hi CD127neg) and memory (CD44hi CD127hi) subsets were sorted on d8 and d60 post-infection, respectively.
Growth protocol
B5 TCR Tg mice (BALB/cJ) were infected with Plasmodium chabaudi.
Extracted molecule
total RNA
Extraction protocol
RNA was isolated and processed according to Agilent Array protocol (PhalanxBio, Inc, San Diego, CA).
Label
Cy3
Label protocol
0.1 µg of total RNA was amplified and labeled using Agilent's Low Input Quick Amp Labeling Kits. Dye incorporation and aRNA yield were checked using the NanoDrop ND-1000 Spectrophotometer.
Hybridization protocol
Cyanine-3 (Cy3)-labeled cDNA of the sample (0.6 µg) was hybridized onto Agilent SurePrint G3 Mouse Gene Expression Microarray (8x60K) using Agilent’s Gene Expression Hybridization Kit. Hybridization was performed for 17h, rotating at a speed of 10rpm at 65? in an Agilent hybridization oven.
Scan protocol
After hybridization, microarrays were washed for 1 minute at room temperature with Wash Buffer 1 and 1 minute at 37? with Wash buffer 2. Microarrays were scanned with Agilent Microarray Scanner and the fluorescence intensities were quantified.
Data processing
Array Raw data files were loaded into R version 2.12.1 to process data analysis.
