Cd117 apc

C57BL/6 mice (n = 4) were injected ip. with PBS (100 μL) or CuNP (200 μg/mouse/100 μL). After 1 h, peritoneal lavage cells were collected and studied by flow cytometry. The cells were stained with several cell surface markers in order to determine the recruitment of different populations. The antibodies used in the analysis were CD11b-PE, Gr1-FITC, F4/80-PerCP Cy5.5, CD19-PE, CD3-FITC, CD4-APC, NK1.1-PerCP Cy5.5, CD117-APC (Biolegend). Samples were acquired with a FACSCalibur flow cytometer (BD Biosciences) and data were analyzed using FlowJo software (Tree Star Inc., USA). The results were expressed as a percentage of live cells (mean ± SE, *p < 0.05 and ****p < 0.0001).

Approximately 100 μl peripheral blood was collected from mice. After lysis of red blood cells, the cells were stained with different antibodies. Lung cells extraction and staining were performed as described previously [22] . We first cut the lungs in 15 ml tubes using small scissors. After cutting, we added cold RPMI 1640 (1 ml/pair of lungs), then placed the tubes on ice. Once all lungs were cut, pre-warmed (37 °C) 2× digestion medium was added to each tube (1 ml/pair of lungs). Next, the tubes were placed in a warm water bath (37 °C) and incubated under shaking for 45 min, followed by adding 10 ml cold MACS buffer was added to each tube. After filtering with a 100 μM cell strainer, osmotic lysis buffer (1 ml/pair of lungs) was added to the tubes and then incubated at room temperature for 2 min. Lastly, the cells were spun down and resuspended in MACS buffer. Antibodies: CD45 (APC-eFlour780), CD3e (Alexa Fluor700), CD5 (eFluor450), CD4 (PE), CD8a (BB515), CD25 (PE-Cy5.5), CD69 (PE-Cyanine7), CD44 (BV605), CD62L (APC), MHCII (FITC), Ly6G (Alexa Fluor700), NK1.1 (BV421), CD19 (PE-Cy5.5), CD11c (PE), CD11b (APC), CD5 (PE-Cy7), Foxp3 (PE) (eBioscience, Ltd., UK), CD4 (Biotin) (BD Pharmingen, San Diego, CA, USA), and CD117 (APC) (BioLegend, Inc, San Diego, CA, USA).