Anti cd11b apc

Single-cell suspensions were stained using mouse-specific Abs, including FITC–anti-CD11b (BD Biosciences), Alexa 647–anti-IL-23p19 (eBioscience), PE–anti-TNF-α, PE/Cy7–anti-Ly6C for macrophages and subsets. For CD4 T cell and subset staining, cells were stained with FITC–anti-CD4, PE/Cy7–anti-Thy1.2, APC–anti-IFN-γ, and PE–anti-IL-17. Intracellular and intranuclear staining was performed as described previously (15 (link)). For macrophages treated with 2-D-gal in vitro, cells were stained with Bio-ICAM followed by streptavidin eFluor 450 (eBioscience) and APC–anti-CD11b, FITC–anti-CD80, and PE–anti-CD86. 2D2 CD4 T cells were identified by staining with APC–anti-CD4, PE/Cy7–Vβ11. DQ-Ovalbumin (DQ-OVA, Invitrogen) was detected in FITC channel. For macrophages loaded with Eα-GFP peptides, cells were stained with bio-YAe (eBioscience), followed by streptavidin eFluor 450 (eBioscience) and APC–anti-CD11b (Biolegend).
To determine the degree of ERK1/2 intracellular signaling, we performed phospho-flow studies according to the protocol from BD Biosciences. Intracellular pERK1/2 was stained with Rabbit anti-ERK1 (T202/Y204)/ERK2 (T185/Y187) (R&D systems), followed by Alexa488–goat anti-rabbit IgG (Invitrogen).
Data were acquired on a BD LSRII flow cytometer and analyzed using FlowJo software (Tree Star, Inc.).

Cell suspensions were collected after 2 days of culture and stained with combinations of the following Abs: APC anti-CD11b (eBioscience), AF700 anti-CD3e, AF750 anti CD4, PerCp-Cy5.5 anti-CD8a, BV421 anti-IgG, AF750 anti-CD19 (Biolegend), PE anti-CD138 (Syndecan-1, R&D Systems), FITC anti-IgA (Southern Biotech). For intracellular detection of IgG and IgA, golgi-stop was added to cell cultures 4 hours before harvest and extracellular staining with anti-CD19 and anti-CD138 Abs. Cells were then fixed and permeabilized before intracellular staining with anti-IgG and anti-IgA Abs. Stained cells were analyzed with an Attune NxT flow cytometer (Thermo Fisher Scientific, Waltham, MA).

Collagenase Type II digested tissues were passed through a 40μm cell strainer. Peripheral blood was prepared by lysing one drop of tail blood for twenty minutes with RBC lysis buffer. Single-cell suspensions were stained with eFluor780- anti-CD45 (eBioscience), PE-anti-Ly6g (BD), APC-anti-Cd11b (eBioscience), FITC-anti-B220 (eBioscience) and eFluor450 anti-F4/80 (eBioscience). Flow cytometry was performed using an LSRFortessa (BD). Flow cytometry data was analyzed using FlowJo software (BD Biosciences).

Murine neutrophils were purified from bone marrows as previously described (51 (link)). Briefly, bone marrow cells collected from mice were treated with the ACK buffer (155 mM NH₄Cl, 10 mM KHCO₃ and 127 μM EDTA) for red blood cell lysis, followed by a discontinuous Percoll density gradient centrifugation. Neutrophils were collected from the band located between 81% and 62% of Percoll. The transient transfection of neutrophils was done as previously described (14 (link), 16 (link), 17 (link), 51 (link)-57 (link)). In brief, three million neutrophils were electroporated with 1.6 μg endotoxin-free plasmids or 300 nM of siRNA using the human monocyte nucleofection kit (Lonza, Switzerland) with an Amaxa electroporation system. The cells were then cultured in the medium supplied with the kit containing 10% FBS and 25 ng/ml recombinant GM-CSF (PeproTech, Rocky Hill, NJ) for periods indicated in the figure legends. Cell sorting was done by a FACS Aria sorter (BD, San Jose, CA). For cell viability analysis, neutrophils were stained with the Zombie Violet™ Fixable Viability dye followed by incubation with APC anti-CD11b (17-0112-82, eBioscience) and PerCP-Cy5.5 anti-Ly6G (560602, BD Biosciences), and analyzed on a BD LSRII flow cytometer.

Isolated liver immune cells and cultured liver macrophages were collected and counted in an automated cell counters (Cellometer Auto X4, Nexelcom Bioscience) after staining for acridine orange (Sigma-aldrich). Antibodies (details in table S2) including BUV396-anti-CD45 (BD bioscience; # 563791), FITC-anti-Ly6G (Biolegend; #127605), APC/Cy7-anti-F4/80 (Biolegend; #123117), PerCP/Cy5.5-anti-Ly6C (Biolegend; #128011), PE/Cy7-anti-CD31 (Biolegend; #102417), Alexa488-anti-iNOS (Thermo Fisher; #53-5920-82), APC-anti-CD11b (Thermo Fisher; 17-0112-82), PE-anti-Tim4 (Thermo Fisher; 12-5866-82), or goat anti-Clec4f (R&D systems; #AF2784) were incubated with FACS buffer (2% fetal bovine serum, 2 mM EDTA, and sodium azide in PBS) on ice for 30 min. In some experiments, the primary cultures of KCs were incubated with 100 ng/ml of biotinylated LPS (Invivogen, #tlrl-lpsbiot) for 2 hours. Then biotin was detected using PE/Cy7-streptividin (Biolegend; #405206). After surface staining of biotin, in some experiments, internalized biotin-LPS was stained using BV605-streptavidin (Biolegend; #405229) in cells permeabilized using the Intracellular Fixation & Permeabilization Buffer Set (Thermo eBioscience; #88-8824). After washing and resuspension, cells were analyzed on a BD FACS Symphony machine and analyzed by FlowJo software (BD biosciences).