Apc rat anti mouse cd11b

The local immune response was characterized by measuring total leucocytes and PMNs in the lavage using flow cytometry (FACSCalibur BD Biosciences, Heidelberg, Germany). For neutrophilic granulocyte characterization, forward and side scatter characteristics and the myeloid cell marker APC rat anti-mouse CD11b (BD Pharmingen, Frankfurt, Germany, monoclonal, catalog number: 553991, clone: A95-1) were used.

As above, ipsilateral hemispheres were processed for flow cytometry (FCM) using antibodies (APC mouse anti-rat CD11b, BD Bioscience; Alexa700 mouse anti-rat CD45, BioLegend; Pacific blue mouse anti-rat granulocytes, BD Bioscience; FITC mouse anti-rat CD163, BioLegend; and PE mouse anti-rat CD86, BioLegend). The cells were analyzed with a Gallios instrument flow cytometer (Beckman Coulter, Tokyo, Japan) and FlowJo software (version 7.6.5, Treestar, Ashland, OR), as described previously (Abe et al., 2018 (link)).

Three rats in each group were randomly selected for quantitative detection of macrophages in the ST36 acupoint tissue. Tissue samples were cut into 1 to 2 mm pieces. Fragments were digested for 1 h at 37°C with 9.75 ml RPMI medium1640/each tissue sample containing 10 mg collagenase, 1 mg hyaluronate, and 0.25 ml HEPES (Sigma-Aldrich, St. Louis, MO, USA). The digested fragments were pressed through a 70 μm nylon cell strainer (BD Falcon, Franklin Lakes, NJ, USA) to remove particles. Macrophage was stained with PE Mouse Anti-Rat CD45, APC Mouse Anti-Rat CD11b (BD Biosciences, San Jose, CA, USA), and FITC Mouse Anti-Rat CD68 (AbD Serotec, Kidlington, UK). Cells stained with isotype-matched irrelevant control antibodies and unstained cells were used as negative controls. Cells were detected by a Attune™ NxT Acoustic Focusing Cytometer (Thermo Fisher Scientific, San Jose, CA, USA) and analyzed by Attune NxT software (Thermo Fisher Scientific).

The density of passage 2 BMSCs was adjusted to 2 × 10⁷ cells/mL. In the single label group, after adding 5 μL of APC mouse anti-rat CD11b (BD, USA), PE-Cy™7 mouse anti-rat CD45 (BD, USA), V450 mouse anti-rat CD29 (BD, USA), PE mouse anti-rat CD90 (BD, USA), and PE mouse anti-rat CD106 (BD, USA) to each flow detection tube, 45 of μL staining buffer was added. For the multi-color labeling group, 5 µL of each of the above antibodies were added to a flow detection tube, followed by 25 μL of staining buffer. For the blank control, only 50 μL of staining buffer was added. Next, 50 μL of cell suspension was added to each tube, mixed well, incubated at 4 °C for 0.5 h protected from light, resuspended, and washed twice with staining buffer. Finally, 500 μL of staining buffer was added to each tube and detected by flow cytometry.