Pe hamster anti mouse cd3e

Samples of 150 μl of blood were collected from the tail vein of the animals at specific time points post-injection (1, 7 and 13 days) and PBMCs were isolated to analyze the immunological impact on them. Briefly, heparinized blood was centrifuged at 1000 × g for 10 min. Serum was separated and kept at −20 °C for further cytokine analyses. Remaining sample was washed with 1 ml PBS and centrifuged at 1500 rpm. Supernatant was then discarded and cells were treated for 30 min in darkness with PE Hamster Anti-Mouse CD3e (BD 553063), FITC Rat Anti-Mouse CD45R/B220 (553087) and PerCP-Cy5.5 Rat Anti-Mouse CD11b (BD 561114) in order to stain T lymphocytes, B lymphocytes and monocytes/macrophages, respectively. Afterwards, samples were centrifuged, supernatant discarded and cells treated with ammonium chloride potassium buffer (ACK) to eliminate red blood cells. After a final centrifugation, supernatant was again discarded and cells were resuspended in PBS before being subjected to flow cytometry analysis. The percentage of cells in each subpopulation was determined by acquiring at least 5,000 events using a FACSCalibur flow cytometer (BD, Franklin Lakes, NJ) and analyzing the data with Flowing Software 2.5.1.

For flow cytometry analysis, whole blood samples were stained following the almost identical protocol already described elsewhere^{72 (link)} by using the following fluorochrome-labeled anti-mouse mAbs: FITC Rat Anti-Mouse CD45; APC Rat Anti-Mouse Ly-6G; PE-CF594 Rat Anti-Mouse Ly-6G and Ly-6C; PE-Cy7 Rat Anti-Mouse CD4; Alexa Fluor 700 Rat Anti-Mouse CD8a; APC-Cy7 Rat Anti-Mouse CD45R; PE Hamster Anti-Mouse CD3e and PerCP-Cy5.5 Rat Anti-Mouse CD335 (BD Biosciences, San Diego, USA). In brief, protected from light, blood was incubated for 15 min at room temperature. The stained samples were then treated with BD FACS lysing solution (BD Biosciences) following manufacturer’s instructions. Thereafter, white blood cells were washed twice with washing buffer (PBS, 1% BSA and 0.1% sodium azide), resuspended in measuring buffer (PBS, 0.1% BSA and 0.1% sodium azide) and measured by flow cytometry using LSR Fortessa (BD Biosciences, San Diego, USA). Plots were compensated and analyzed by FlowLogic Software 7.2.1 (Inivai Technologie, Mentone Victoria, Australia). Gating strategy is shown in Supplemental Fig. 1.