1% saponin, 0.2% NaN3), followed by staining with αIL-7-biotin and streptavidin-APC.
Samples were measured and analyzed as described in “Antibodies and flow cytometry”. Single-cell suspensions of naïve CD45.1+ splenocytes were prepared, and erythrocytes were removed. Half of the cells were pulsed with gp33 (10−6 M) at 37°C for 90 min. Then, the cells were washed twice with PBS, adjusted Tanespimycin concentration to 2×106 cells/mL, and labeled with CFSE (Molecular Probes, Eugene, OR, USA) at either a final concentration of 5 μM (gp33-pulsed splenocytes, CFSE high) or of 0.1 μM (unpulsed splenocytes, CFSE low) for 10 min at 37°C. After labeling, FCS was added up to a final concentration of 10%, and cells were washed with PBS at 4°C. Briefly, 3×107 CFSE-labeled, gp33-pulsed and 3×107 CFSE-labeled, unpulsed CD45.1+ splenocytes were selleck compound injected i.v. into H8-CML mice, αCD8-treated H8-CML mice, naïve C57BL/6 and LCMV-immune mice which had been infected i.v. with 200 pfu LCMV-WE 8 wk previously. After 8, 24 and 48 h, blood was collected, and the reduction of the CFSE high population normalized to the CFSE
low population was calculated by flow cytometry analysis. P14×CD45.1 T cells were isolated and purified by MACS (Miltenyi Biotec) for CD8+Va2+ T cells. In total, 2.5−4×106 CD8+Va2+CD45.1+ cells were injected i.v. into H8-CML mice, H8×IL-7−/−-CML mice, naïve C57BL/6 control mice and C57BL/6 mice chronically infected with 107 pfu LCMV Docile (all recipient mice were CD45.1−). CML disease progression and expansion of transferred CD8+Va2+ T cells were monitored GPX6 by FACS analysis of blood and spleen. For isolation of total spleen mRNA, 30 mg of tissue were frozen in liquid nitrogen and homogenized using a stainless steel bead and tissue lyser (Qiagen, Hombrechtikon, Switzerland), followed by RNA extraction (RNeasy
mini kit, Qiagen). For isolation of granulocyte mRNA, single-cell suspensions of naïve C57BL/6 or CML spleens were sorted for 1.5×106 granulocytes or GFP+ granulocytes, respectively, into RNAprotect® cell reagent (Qiagen) on a FACS Aria unit (BD Biosciences). RNA was extracted and its concentration was determined by spectrophotometry (Nanodrop ND-1000, Witec AG, Littau, Switzerland). Reverse transcription was performed using 0.25–1 μg of mRNA, random oligonucleotides and AMV-RT (Roche, Basel, Switzerland). For conventional RT-PCR, we used Taq-Polymerase (Roche) and the following primers: β-actin sense 5′-TGGAATCCTGTGGCATCCATGAAA-3′, β-actin antisense 5′-TAAAACGCAGTCCAGTAACAGTCCG-3′, IL-7 sense 5′-GGAATTCCTCCACTGATCCT-3′, IL-7 antisense 5′-CTCTCAGTAGTCTCTTTAGG-3′ (Microsynth, Balgach, Switzerland). For quantitative real-time RT-PCR, we used 10 ng of cDNA per well, TaqMan® Universal PCR Master Mix and TaqMan® Gene Expression Assays for IL-7 (Mm00434291_m1) and the four housekeeping genes GAPDH (Mm99999915_g1), β-actin (Mm00607939_s1), β-Glucuronidase (Mm00446957_m1) and Transferrin-Receptor (Mm00441941_m1) (Applied Biosystems, Rotkreuz, Switzerland).