CLINICS 2012;67(7):805-813 FTY720 treatment in skin allograft rejection Commodaro AG et al. Figure 2 - Evaluation of the effect of transplantation and FTY720 treatment on draining lymph nodes (axillary) five days after F1 skin graft transplantation in the C57BL/6 mice. A - FTY720 treatment reduces the number of lymph node cells (p = 0.022). B - FTY720 treatment reduces the percentage of cells (p = 0.028) with the Treg phenotype (CD4+Foxp3+). The results are expressed as the means ¡ SD. skin transplantation, respectively. Skin from the donor tail 2 (1.0 cm ) was placed on the C57BL/6 mice and fixed with 6- 0 nylon sutures (Brasuture, Brazil) at each corner. The recipient mice were either transplanted only (Tx, n = 5) or transplanted and submitted to the daily administration of FTY720 (Novartis, Switzerland; 1 mg/kg/day diluted in distilled water) by gavage for 21 days, starting three days before the transplantation (Tx + FTY, n = 5). The grafts were observed daily to establish skin allograft survival (rejec- tion = 90% skin necrosis). In another set of experiments, the immunological evaluation was performed five days post- transplantation. Experimental design 1) To evaluate skin allograft survival, the following steps were completed: FTY720 treatment, skin transplantation, and graft follow-up. 2) An immunological evaluation of C57BL/6-transplanted mice either treated or not treated with FTY720 was performed. Immunological evaluation Five days post-transplantation, the spleens, axillary lymph nodes (ALNs) and skin allografts were harvested from the recipient mice. Single-cell suspensions from the spleen, ALNs and skin were counted in a Neubauer chamber. The spleen cells were used for in vitro stimulation and qPCR analysis, whereas the ALNs and graft-infiltrating cells were evaluated by flow cytometry. Flow cytometry Cell suspensions (16106) from the ALNs and grafts were incubated for 30 min with rat anti-mouse monoclonal anti- body CD4PerCP-Cy5.5 (eBioscience, San Diego, CA, USA). After this period, the cells were washed with PBS, fixed for 15 min with 1% paraformaldehyde, permeabilized for 6 min with 0.2% Triton X-100 and stained with rat anti-mouse Foxp3 FITC for 30 min (eBioscience, San Diego, CA, USA). The stained cells were acquired in a FACSCalibur Flow Cytometer (BD Biosciences, San Jose, CA, USA), and the data were analyzed using Cell Quest Pro software (BD Biosciences). At least 30,000 events were acquired for each analysis. Quantitative analysis of gene expression by real- time PCR The total RNA from the skin graft and spleen cells was extracted with Trizol (Invitrogen Life Technologies) according to the manufacturer’s instructions and reverse-transcribed into complementary DNA with Superscript III (Invitrogen Life Technologies). For the real-time PCR of the IL-10, IL-6, FOXP3, IL-23 and IL-27 genes, 0.5 mL of primer, 1 mL of cDNA, 3.5 mL of RNAse/DNAse-free water and 5 mL of Taqman Fast Universal Master Mix (Applied BiosystemsH) were used. The PCR was performed in an Applied Biosystems 7900HT Fast Real-Time PCR SystemH. The mRNA level was determined following normalization to b-actin. We performed qPCR in the spleen for IL-10, IL-6, FOXP3, IL-23 and IL-27. In the skin graft, we performed qPCR for IL-10, FOXP3, IL-23 and IL-27. Bio-Plex The spleen cells were harvested at day five post-skin transplantation and stimulated in vitro with anti-CD3. A Bio-Plex mouse cytokine assay kit (Bio-Rad Laboratories, Hercules, CA, USA) was used to test the samples for the presence of the specific cytokines IL-10, IL-6 and IL-17. The assay was performed in the Bio-Plex suspension array system, and the data were analyzed using Bio-Plex Manager software version 4.0. The standard curves ranged from 1.95 to 32 000 pg/ml. 807