Apc anti mouse cd11c

In order to examine activation status of the cells, BMDCs were treated with Duolac ATP or LPS for 24 h at 37°C. The cells were stained with anti-mouse CD86-FITC, PD-L1-PE, MHC II-PE-cy7, CD11c-APC (all from BD Biosciences) for 20 min at 4°C in the dark. To test the increase of Treg, CTV labeled CD4⁺ T cells cultured with Duolac-treated BMDCs for 3 days were stained with anti-mouse CD4-PE (BD Biosciences). After surface staining, CD4⁺ T cells were fixed and stained with anti-mouse Foxp3-APC mAb (BioLegend, Dedham, MA, United States) using FOXP3 Fix/Perm Buffer Set (BioLegend). In vivo examination, single cells from mLNs and PP were isolated from AD mice. Population changes of DCs and Tregs were examined as aforementioned. To analyze for subpopulation of Th cells, total mLN and PP cells were stimulated with PMA and ionomycin (Sigma-Aldrich) in the presence of brefeldin A for 4 h. After the stimulation, the cells were stained with appropriate combination of anti-mouse CD11c-APC, CD4-bv605, Foxp3-APC, IFN-gamma-PE, IL-4-bv605, and IL-17-APC-cy7 mAb (all from Biolegend). The cells were washed and the expression was examined using a FACSCanto II (BD Biosciences). All flow cytometric data acquired were analyzed with FlowJo software (Tree Star, Ashland, OR, United States).

Inserts from 24-well transwell plates (Costar, Sigma-Aldrich) were coated with collagen type 1 (Advanced Biomatrix) and fibronectin (Merck Millipore) (10 μg/ml each) and seeded with 175'000 imLECs in imLEC medium on the lower side or on the upper side of the transwell membrane, in analogy to the setup originally described by Johnson et al. (52 (link)). The medium was exchanged after 6 h and again the next day. Two days later, 1 × 10⁵ LPS-activated BM-DCs (WT or ALCAM^−/−) were added to the top well and left to migrate toward the bottom well containing 100 ng/ml of CCL21 (PeproTech) in DMEM/F12 medium containing 2% FCS. After 4 h, transmigrated DCs were stained with anti-mouse CD11c-APC (BioLegend) and quantified by FACS. For assessing antibody efficacy, WT DCs and LECs were both pre-incubated with I/F8-Fc or KSF-Fc (10 μg/ml) for 30 min at 37°C prior to performing the experiment in presence of antibodies.

Anti-PD-L1 (ab213480, Abcam), anti-GAPDH antibody (2118, CST), anti-Phospho-TBK1/NAK antibody (5483, CST), HRP-conjugated secondary antibodies (7074, CST), anti-PD-L1(BE0101, BioXcell), anti-mouse CD16/32 antibody (553,141, BD Pharmingen), Fixable Viability Stain (565,388, BD Pharmingen), anti-CD80-BV421(562,611, BD Pharmingen), anti-CD86-PE-Cy7(560,582, BD Pharmingen), anti-mouse PD-1-PE (135,206, BioLegend), anti-mouse LAG3-PE-Cy7 (125,226, BioLegend), anti-mouse TIM3-BV421 (119,723, BioLegend), anti-mouse FOXP3-BV421 (126,419, BioLegend), anti-mouse Ki67-PE-CY7 (652,426, BioLegend), anti-mouse PD-L1-PE (124,308, BioLegend), anti-mouse CD11b-FITC (101,206, BioLegend), anti-mouse Ly6G-PE (551,461, BD Pharmingen), anti-mouse Ly6C-PE-Cy7 (128,018, BioLegend), anti-mouse F4/80-BV421 (123,132, BioLegend), anti-mouse CD206-APC (141,708, BioLegend), anti-mouse TNFα-PE (554,419, BD Pharmingen), anti-mouse CD3-BV605 (100,237, BioLegend), anti-mouse CD11C-APC (117,310, BioLegend), anti-mouse MHC-II-BV421 (107,632, BioLegend), anti-mouse NK1.1-PE-Cy7 (552,878, BD Pharmingen), anti-mouse CD80-PE (552,769, BD Pharmingen).

The expression of CD11b, CD206, CD11c, and F4/80 in BMSC-derived macrophages were analyzed using the FACS Calibur (Becton Dickinson, United States) and the CellQuest software (Becton Dickinson). The adherent cells were washed with PBS and collected using Accutase (Nacalai Tesque, Kyoto, Japan) and resuspended in 50 μL staining buffer (BD Pharmingen, Franklin Lakes, NJ, United States). The harvested cells were blocked with 2 μL Human Trustain FcX (Fc receptor Blocking Solution; BioLegend) for 10 min at room temperature, and stained with 2 μL antibodies in the dark for 30 min at 4°C. FITC anti- mouse CD11b, FITC anti-human CD11b, Alexa Fluor^®488 anti-human CD11b, BV421 anti-mouse CD206, PE anti-mouse F4/80 and APC anti-mouse CD11c (BioLegend).

At 7 dpi, pulmonary single-cell suspension was obtained and labeled using the method described above but with the following labeling antibodies: APC anti-mouse CD11c, FITC anti-mouse MHC Class II, PE anti-mouse NKp46, PE/Cy7 anti-mouse CD19, PerCP/Cy5.5 anti-mouse CD3ε, PE anti-mouse F4/80, PE/Cy7 anti-mouse Ly-6G, PerCP/Cy5.5 anti-mouse Siglec H (all from BioLegend, USA), FITC anti-mouse CD4 and PE anti-mouse CD8a (both from eBioscience).

Chicken anti-GFP antibody (Abcam, ab13970, 1:500), Rabbit anti-Prospc (Millipore, ab3786, 1:500), Rat anti-Ki67 (ebioscience, 14-5698-82, 1:200), Rat anti-F4/80 (BioRad, MCA497GA, 1:200), APC anti-mouse CD326 (BioLegend, catalog#118214), PE/Cyanine7 anti-mouse CD45 (BioLegend, catalog#103114), PE/Cyanine7 anti-mouse CD31 (BioLegend, catalog#102418), PE anti-mouse F4/80 (BioLegend, catalog#123110), APC anti-mouse CD11c (BioLegend, catalog#117310), PerCP-Cyanine5.5 anti-human/mouse CD11b (Tonbo, catalog#65-0112), PE anti-mouse FOXP3 (BD biosciences, catalog#560408), APC anti-mouse CD4 (BD biosciences, catalog#553051), FITC Mouse IgG1 isotype control (BD biosciences, catalog#555748), PE Mouse IgG1 isotype control (BD biosciences, catalog#555749), APC Mouse IgG1 isotype control (BD biosciences, catalog#555751), FITC anti-human CD90 (BD biosciences, catalog#555595), PE anti-human CD73 (BD biosciences, catalog#550257), APC anti-human CD105 (BD biosciences, catalog#562408), FITC anti-human CD45 (BD biosciences, catalog#555482), PE anti-human CD34 (BD biosciences, 550761), Alexa Fluor 488 Donkey anti Chicken (Jackson Immuno Research, catalog#703-545-155), Alexa Fluor Cy3 Donkey anti Rat (Jackson Immuno Research, catalog#712-165-153), and Alexa Fluor 488 Donkey anti Rabbit (Jackson Immuno Research, catalog#711-545-152).

Flow cytometry was performed as previously reported (7 (link)). Briefly, after perfusion of the kidneys with PBS, 1 kidney was removed, minced into fragments, and digested in RPMI 1640 containing 2 mg/ml collagenase type D and 100 μg/ml DNase I for 45 minutes at 37°C, with intermittent agitation. Kidney fragments were passed through a 40 μm mesh (Falcon; BD Biosciences), yielding single-cell suspensions. Cells were centrifuged (300g, 10 minutes, 4°C), resuspended in FACS buffer, kept on ice, and counted using a Bio-Rad TC20 automated cell counter. 10⁵ cells were incubated in 2.5 μg/ml Fc blocking solution and stained for 60 minutes at 4°C with antibodies, including FITC rat anti-mouse CD45, APC anti-Ly6G, PE/Cy7 anti-mouse F4/80, Pacific Blue anti-mouse CD11b, APC anti-mouse CD11c, or isotype control (all BioLegend). Cells were then washed and stained for 15 minutes at room temperature with annexin V or H₂DCFDA and resuspended in 1× annexin binding buffer (or PBS with 1% BSA for H₂DCFDA). For cell cycle analysis, cells were stained with propidium iodide at 50 μg/ml. After immunostaining, cells were analyzed immediately on a Novocyte flow cytometer with NovoExpress Software (Acea Biosciences) for data acquisition, and data analysis was performed using FlowJo v10 software (Tree Star).