Fitc conjugated rat anti mouse cd3

For the simultaneous detection of cell surface expressed CD4, CD8 and CD3, a triple labelling technique was used. Thymocyte samples were incubated with monoclonal antibody cocktails for 30 min in 100 ml binding buffer on ice (PBS containing 0.1% NaN₃ and 0.1% BSA), then washed twice in PBS, and finally resuspended in 500 ml 0.1% buffered PFA (paraformaldehyde) in PBS. For staining the following monoclonal antibodies were used: phycoerythrin (PE) conjugated rat anti-mouse CD4, CyChrome (CyC) conjugated rat anti-mouse CD8 and FITC conjugated rat anti-mouse CD3, all purchased from BD Pharmingen, CA. Samples were measured and analysed in a FACSCalibur flow-cytometer (Becton Dickinson, San Jose, CA), using the CellQuest software. Generally 10.000 events were recorded. Thymocytes were gated according to their size and granularity on forward and side scatter dot plots. The gate set on untreated control living thymocytes was used for the analysis of all samples. We used fluorescent dot plots for both comparing the different samples and for calculating the ratio of positively stained cells.

A dose of 1 μg of Lpl1(+sp) in 10 μl of PBS or PBS alone as internal control, were s.c. injected into the auricle of anesthetized C57BL/6 wild-type (n = 6), and TLR2^−/− (n = 5) mice. On day 1 after injection, the ear tissues were collected, placed in RPMI medium (Fisher Scientific), and subjected to enzymatic digestion with 4 mg/ml Collagenase IV (Fisher Scientific) and 0.4 mg/ml DNase I (Sigma-Aldrich) in RPMI medium, followed by incubation for 1 h at 37 °C with shaking. A single-cell suspension was obtained after the tissue was homogenized and passed through a 40 µm cell strainer (Becton Dickinson). Skin tissue cells were then analyzed using the following antibodies: APC-R700-conjugated anti-mouse CD45 (BD Biosciences), V450-conjugated anti-mouse CD11b (BD Biosciences), APC-conjugated anti-mouse F4/80 (BioLegend), PE-Cy7-conjugated anti-mouse Ly-6G (BD Biosciences), BV605-conjugated anti-mouse Ly-6C (BD Biosciences), PerCP-Cy5.5-conjugated anti-mouse CD335 (BD Biosciences), and FITC-conjugated rat anti-mouse CD3 (BD Biosciences). Cells were acquired on a BD FACSLyric flow cytometer (BD Biosciences) and data were analyzed using FlowJo version 10.1 software (Tree Star, Ashland, USA).