RNA was extracted from lungs of control and infected mice with Trizol reagent (Invitrogen), transcribed to cDNA, and primers for CCL2 (F 5-TGGCTCAGCCAGATGCAGT-3, R 5-TTGGGATCATCTTGCTGGTG-3), CCL4 (F 5-TCTTGCTCGTGGCTGCCT-3, R 5-GGGAGGGTCAGAGCCCA-3), CCL5 (F 5-CAAGTGCTCCAATCTTGCAGTC-3, R 5-TTCTCTGGGTTGGCACACAC), CCL17 (F 5-CAGGGATGCCATCGTGTTTC-3, R 5-CACCAATCTGATGGCCTTCTT-3), CCL22 (F 5-TACATCCGTCACCCTCTGCC-3, R 5-CGGTTATCAAAACAACGCCAG-3), CXCL9 (F 5-AATGCACGATGCTCCTGCA-3, R 5-GGTCTTTGAGGGATTTGTAGTG-3) and CXCL10 (F 5-GCCGTCATTTTCTGCCTCA-3, R 5-CGTCCTTGCGAGAGGGATC-3) (Integrated DNA Technologies) used to quantify gene expression by PCR.
DNA from lungs of PbN-infected mice was extracted by illustra tissue & cells genomicPrep Mini spin kit (GE Healthcare). The primers of mouse endogenous gene, GAPDH (Forward: GGCAAATTCAACGGCACAGT and Reverse: AGATGGTGATGGGCTTCCC) and the Plasmodium berghei 18 s ribosomal 18 s gene (Forward: AAGCATTAAATAAAGCGAATACATCCTTA and 18 s Reverse: GGAGATTGGTTTTGACGTTTATGT) were used to determine the parasite load in the lungs by relative quantification, normalized with the mouse GAPDH (∆CT). The quantitative PCRs were carried out with the Platinum SYBR Green protocol (Invitrogen) on an Applied Biosystems 7500 real-time PCR system.
DNA from lungs of PbN-infected mice was extracted by illustra tissue & cells genomicPrep Mini spin kit (GE Healthcare). The primers of mouse endogenous gene, GAPDH (Forward: GGCAAATTCAACGGCACAGT and Reverse: AGATGGTGATGGGCTTCCC) and the Plasmodium berghei 18 s ribosomal 18 s gene (Forward: AAGCATTAAATAAAGCGAATACATCCTTA and 18 s Reverse: GGAGATTGGTTTTGACGTTTATGT) were used to determine the parasite load in the lungs by relative quantification, normalized with the mouse GAPDH (∆CT). The quantitative PCRs were carried out with the Platinum SYBR Green protocol (Invitrogen) on an Applied Biosystems 7500 real-time PCR system.