Anti cd68 apc

Three milliliters of PBMCs with a concentration of 5 × 10⁶ cells/ml was cultured in a well of a 6-well plate. After 1 h of incubation at 37°C and 5% CO₂, the nonadherent cells were washed 3 times with warm PBS. The remaining adherent monocytes were prestimulated for 24 h with concentrations of different ligands that would induce either training or tolerance, such as β-glucan (1 μg/ml), MDP (1 μg/ml), flagellin (1 μg/ml), R848 (1 μg/ml), Pam3CSK4 (1 μg/ml), and RPMI (negative control). The stimuli were washed away and the cells were further incubated for 5 days in the presence of RPMI supplemented with 10% pooled human serum. On day 7, the cells were incubated for 1 h with Versene solution, collected, harvested by centrifugation, and suspended in PBS supplemented with 1% protein blocking agent (PBA). The cells were washed two times and stained extracellularly with the following antibodies: anti-CD45-PeCy7 (Beckman Coulter), anti-CD68-APC (BioLegend), anti-CD14-PE (Beckman Coulter), anti-CD11b-FITC (Beckman Coulter), and anti-DC-SIGN-APC (BioLegend). The samples were measured on a fluorescence-activated cell sorter (FACS) FC500, and the data were analyzed using the CXP software (Beckman Coulter).

Monocytes from BALB/c spleen samples were sorted using a Mouse Monocyte Isolation Kit (Miltenyi), co-cultured with Tregs at monocyte:Treg ratios of 2:0 and 2:1, and exposed to different stimuli to determine the effect of a miR-146a deficiency in Tregs on macrophage polarization. Macrophage type 1 (M1) cells were stimulated with lipopolysaccharide (LPS; 10 µg/ml; Solarbio) + interferon (IFN)-γ (20 ng/ml; PeproTech), and macrophage type 2 (M2) cells were stimulated with LPS (10 µg/ml; Solarbio)+IL-4 (50 ng/ml; PeproTech). After 72 h of co-culture, the cells from each group were harvested and labeled with anti-CD16/32-FITC (Catalog No.: 101305; BioLegend, USA), anti-CD206-FITC (Catalog No.: 141703; BioLegend, USA), and anti-CD68-APC (Catalog No.: 137007; BioLegend, USA) antibodies, and the ratios of various types of cells were detected using flow cytometry.
The levels of tumor necrosis factor (TNF)-α, IL-1β, and IL-10 in the supernatant of co-cultured monocytes and Tregs were determined using ELISA kits (DAKEWE) according to the manufacturer’s instructions.

The following antibodies were used for immunoblotting, immunofluorescence, and flow cytometry analysis: anti-Caspase-1 (ab179515; Abcam), anti-NLRP3 (ab263899; Abcam), anti-ASC (sc514414; Santa Cruz), anti-NF-κB P50 (ab32360; Abcam), anti-NF-κB P65 (cst6956; Cell Signaling Technology), anti-phospho–NF-κB P65 (cst3033; Cell Signaling Technology), anti-MyD88 (ab219413; Abcam), anti-IKK-β (ab124951; Abcam), anti-TRAF6 (ab33915; Abcam), anti-GPR81 (ab106942; Abcam), anti-tubulin (11224; Proteintech), anti-pro-IL-1β (cst12242; Cell Signaling Technology), anti–F4/80-PE (123110; BioLegend), anti–CD11b-BV421 (101236; BioLegend), anti–CD68-APC (137007; BioLegend), anti–CD206-AF647 (141712; BioLegend), anti–Ly6G-FITC (11–9668-80; eBioscience), anti-CD16/32 (14–0161-82; eBioscience), anti-CD11b (2185-1-ap; Proteintech), anti-F4/80 (cst70076; Cell Signaling Technology), anti-Ly6G monoclonal antibody (65140-1-ig; Proteintech).
The following reagents were used for this study: caerulein (C9026-1 MG; Sigma, Munich, Germany), ELISA kit for IL-1β, IL-6 and TNF-α (Lianke), alpha-amylase determination kit (BIOSINO, Beijing, China), PBMC isolation kit (LTS1092; TBD), LPS from Escherichia coli O55:B5 (Sigma, Germany), L-lactate assay kit (ab65330; Abcam), sodium L-lactate (L7022; Sigma), L-lactic acid (L1750; Sigma).

The spleens were collected from the recipients in each group, ground, and filtered through a 40-mesh screen. Red blood cells were lysed with red blood cell lysis buffer (Solarbio), and then the remaining cells were washed twice and resuspended in phosphate-buffered saline (PBS; Solarbio). Fluorescent dye-conjugated antibodies were added to 10⁵ cells in 100 µl of PBS and incubated in the dark for 30 min. The following antibodies were used for the flow cytometry analysis: anti-CD4-FITC (Catalog No.: 100405; BioLegend, USA), anti-Foxp3-PE (Catalog No.: 126403; BioLegend, USA), anti-CD16/32-FITC (Catalog No.: 101305; BioLegend, USA), anti-CD206-FITC (Catalog No.: 141703; BioLegend, USA), and anti-CD68-APC (Catalog No.: 137007; BioLegend, USA) antibodies.
Serum levels of interleukin (IL)−10 and transforming growth factor (TGF)-β1 in recipients were determined using ELISA kits (DAKEWE, Beijing, China) according to the manufacturer’s instructions.

Disaggregated single cells isolated from synovial biopsies were treated with GPX4 inhibitor RSL3 for 6 h before staining. Antibodies used were PerCP anti-CD3 (Biolegend, 317338), APC/Cyanine7 CD31 (Biolegend, 303120), APC anti-CD68 (Biolegend, 333810), PE/Cyanine7 anti-CD19 (Biolegend, 302216), anti-FAPα (Invitrogen, BMS168), PE Goat anti-mouse IgG (Biolegend, 405307). Cell death was analyzed by SYTOX Green (Invitrogen) staining. The Fluorescence Minus One (FMO) controls were used to determine the cut-off point between background fluorescence and positive populations. The data was acquired with a BD Fortessa flow cytometer with BD FACSDiVa™ software and analyzed by FlowJo, v.10.5.3.

BMDMs were prepared as described (22 (link)). For erythrophagocytosis assays, macrophages were plated in 12-well culture plates. Refrigerator-stored 7–9 days erythrocytes from control and VFEpoR mice were added (macrophage/erythrocyte ratio was about 1:10) into macrophages. Following 6 hours of incubation, the macrophages were thoroughly washed with PBS 3 times and then analyzed by flow cytometry or Western blot.
For erythrophagocytosis assay using human RBCs, peripheral blood samples were obtained from patients with JAK2^V617F or matched healthy controls. RBC-rich fractions were obtained with High Efficiency Leukocyte Reduction Filter (Haemonetics Corporation, NEO1). Human peripheral monocyte-derived macrophages generated from healthy donors were used for the assays and plated in 24-well, no-tissue culture plates as previously reported (22 (link)). RBCs were added and incubated overnight (macrophage/RBC ratio was about 1:10). After washing for 3 times with PBS to remove the free RBCs, the RBCs were labeled with PerCP/cyanine 5.5 anti-CD235a (BioLegend, 349110) and macrophages were stained with APC anti-CD68 (BioLegend, 333810). Lipid peroxidation was measured by the C11-BODIPY, and cell death was determined by propidium iodide staining.

Cells were stained with the following monoclonal anti-human antibodies: fluorescein isothiocyanate (FITC) anti-M-CSF1-R (R&D Systems, Minneapolis, MN, USA), allophycocyanin-H7 (APC-H7) anti-CD45, PerCP 5.5 anti-HLA-DRII, PerCP 5.5 anti-CD19, Pacific Blue anti-CD3, Alexa Fluor 647 (APC) anti-CD206, (all from Becton Dickinson, Milan, Italy), FITC anti-CD3 (Beckman and Coulter, Marseille, France), APC anti-CD68 (Biolegend, San Diego, California), phycoerythrin (PE) anti-CD68 (eBioscience, San Diego, CA), FITC anti-IL-6 (Miltenyi Biotec, Bologna Italia), PeCy7 anti-CD163 (Biolegend), PE anti-IL-34 (R&D Systems). In all experiments, appropriate isotype control IgG was used. All antibodies were used at 1:50 final dilution. For intracellular staining (CD68, IL-34, IL-6), cells were fixed and permeabilized using IC Fixation buffer and the permeabilization buffer (both from eBioscience) according to the manufacturer’s instruction. Cells were analyzed by flow cytometry (FACSVerse, Becton Dickinson), and analysis was performed by acquiring 10,000 events in the gate of CD45 + live cells according to LIVE/DEAD staining (Life Technologies, Milan, Italy). Flow-cytometry data were analyzed by FlowJo ver. 10.7 (Becton Dickinson).