Histological sections were created from paraffin embedded adipose tissues. Adipocyte size was measured by 200 individual cell diameters being calculated from 10 representative fields viewed at the 10× magnification level with 20 adjacent cell diameters recorded per field. Average cell diameter was calculated and reported. Immunohistochemistry stains were applied to identify classically activated M1 macrophages (pan-macrophage marker CD68 [AbD Serotac Bio-Rad, Raleigh NC), and alternatively (M2) activated macrophages (CD163 [Thermofisher, Rockford IL). CD68 positive cell staining was counted in triplicate, across 15 fields at 40× magnification and all perivascular smooth muscle cells were excluded. CD163 positive cell staining was infrequent and so cells were manually counted from the entire biopsy sample and normalized to the total area of the biopsy piece. A ratio of M2/M1 measurements were calculated and reported. Identification of these cell types is complex, and markers chosen here represent a basic delineation of macrophages, and CD163 staining alters in concert with changes in the metabolic status of people (16 (link)). All histological assessments were conducted by persons blinded to the identity of the samples.
Cd163
Manufactured by Thermo Fisher Scientific
Sourced in United States, China, Germany
CD163 is a transmembrane glycoprotein that functions as a scavenger receptor, primarily expressed on the surface of monocytes and macrophages. It plays a role in the clearance of hemoglobin-haptoglobin complexes from the bloodstream.
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Lab products found in correlation
Cd206, by BioLegend (4 mentions)
Cd206, by BD (3 mentions)
Ifn γ, by Thermo Fisher Scientific (3 mentions)
F4 80, by BioLegend (3 mentions)
Il 10, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Cd206, by Thermo Fisher Scientific (2 mentions)
F4 80, by Thermo Fisher Scientific (2 mentions)
Cd11b, by BD (2 mentions)
Tcrγδ, by Thermo Fisher Scientific (2 mentions)
Live dead fixable blue viability dye, by Thermo Fisher Scientific (2 mentions)
Lsr fortessa 1 flow cytometer, by BD (2 mentions)
Cd16 cd32 93, by BioLegend (2 mentions)
Il 17a, by BioLegend (2 mentions)
Facsdiva, by BD (2 mentions)
Siglec f, by BD (2 mentions)
Alexa fluor 546, by Thermo Fisher Scientific (2 mentions)
Superscript 3 rt kit, by Thermo Fisher Scientific (1 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions)
33 protocols using cd163
1
Adipocyte and Macrophage Characterization
2
Priming Human PBMCs with AngII and LPS
3
Monocyte-Derived Macrophage Polarization
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Anti-CD14 magnetic beads (Miltenyi, Bergisch Gladbach, Germany) were used to isolate CD14+ monocytes from leukocyte cones. Monocytes were polarized to M1 (5 ng/mL hu-GMCSF) or to M2 (25 ng/mL hu-MCSF) (both Peprotech, Cranbury, NJ, USA) macrophages in RPMI-1640 + 1% GlutaMAX + 10% FBS (Gibco) for 7 days. The purity of CD14+ isolation and the phenotype of M1 and M2 macrophages were evaluated by flow cytometry. The following antibodies were used in the study: CD14 (FITC), CD68 (PE-TxRed), CD163 (APC), CD206 (PE-CY7), and S1PR1 (APC) (Thermo Fisher, Waltham, MA, USA). ELISA Duoset kits (R&D Systems, Minneapolis, MN, USA) were used to measure levels of IL-10 and IL-12(p70) released by macrophages.
4
Placental Hofbauer Cell Characterization
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Hofbauer cells isolated from control placentas were plated at a density of 1 × 106 cells/ml in 6-well culture dishes (3 ml total volume). On day 3 post-isolation, cells were serum-starved for 12 h and thereafter switched to complete MaM containing either 25 mM d -glucose (Sigma) to mimic maternal and fetal hyperglycemia, 10 nM Insulin (Calbiochem) to mimic fetal hyperinsulinemia in response to maternal GDM, or a combination of both. Equimolar l -glucose (Sigma) was used as osmatic control, an untreated control grown in MaM only was included. Cells were cultivated for 72 h, receiving treatment every 24 h. Cells were harvested and lysed using RIPA buffer. Protein content was measured using bichinonic acid method (BCA assay, Pierce). 7.5 µg of protein was subjected to electrophoresis (4–20% Mini-Protean TGX gels, Biorad) and blotted onto nitrocellulose membranes (Trans-Blot Turbo System, Biorad). Membranes were incubated with antibodies against CD163 (Thermo Scientific), CD86 and CD209 (both NovusBio) and β-Actin as loading control (abcam); secondary antibodies against mouse and rabbit IgG were from Biorad. Detection was carried out using West Femto ECL substrate (Pierce) on a ChemiDoc XRS system (Biorad).
5
Adipocyte and Macrophage Characterization
6
Multiparametric Immunofluorescence Analysis of FFPE Tissue
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Whole tissue sections from LC03 and LC07 (4‐μm‐thick formalin‐fixed, paraffin‐embedded) were stained with primary antibodies (EpCAM (#2929, CST, 1:500), CD163(#93498, CST, 1:200), Topoisomerase IIα (#12286, CST,1:400), TRIM29(17542‐1‐AP, Proteintech, 1:100), DKK1(Abcam, ab109416, 1:800) sequentially and paired with TSA 7‐colour kit (abs50015‐100T, Absinbio). The order of antibodies/fluorescent dyes was showed as following: anti‐ EpCAM/TSA 480, anti‐ CD163/TSA 780, anti‐TOPIIα/TSA 620, anti‐TRIM29/TSA 520, anti‐DKK1/TSA 570 and then by staining with DAPI (D1306; Thermo Fisher). Pictures were scanned with Aperio Versa 8 tissue imaging system (Leica). Images were analysed using Indica Halo software.
7
Protein Expression Analysis via Western Blot
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Proteins were collected in a RIPA buffer (R0010, Solarbio, China) and separated by 12% SDS‒PAGE (Bio-Rad, China). After being transferred to PVDF membranes, blocked with 5% milk, the membranes were incubated with appropriate primary antibodies overnight at 4 °C. The primary antibodies included CTSK (1:800, #DF6614, Affinity, China), CD68 (1:1000, #14-0688-82, ThermoFisher), CD163 (1:1000, #14-1639-82, ThermoFisher), CD200R (1:1000, #DF4715, Affinity), p-p65 NF-κB (1:1000, #3039, Cell Signaling Technology), p65 NF-κB (1:1000, #3034, Cell Signaling Technology), and GAPDH rabbit antibody (1:1000, #5174, Cell Signaling Technology). After washing with TBS-0.01% Tween 20, the membranes were incubated with secondary antibody (1:1000, #14708, Cell Signaling Technology) for 2 h at 25 °C. After washing, the blots were visualized using an enhanced chemiluminescence reagent (D085075, Bio-Rad, China).
8
Macrophage Characterization and Polarization
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Single-cell suspensions of NR8383 cells and BMDMs were prepared as previously described. BMDMs were incubated first with CD16/CD32 (Thermo Fisher Scientific) and then with FITC-CD68 (MA5-28262, Thermo Fisher Scientific) and PERCPEF710-CD11b/c (46-0110-82, Thermo Fisher Scientific). The percentage of double-positive cells indicates the purity of macrophages. To identify macrophage polarization, cells were first incubated with CD16/CD32, followed by PE-CD86 (12-0860-83, Thermo Fisher Scientific) and CD163 (PA5-78961, Thermo Fisher Scientific). Finally, the cells were incubated in the dark for 1 h with APC (A-10931, Thermo Fisher Scientific). Apoptosis was determined by an Annexin V-FITC/PI double staining cell apoptosis detection kit (Sevenbio, Beijing, China) following the manufacturer’s instructions. Each sample was analyzed by flow cytometry and FlowJo v.10 software.
9
Flow Cytometric Analysis of Sputum Cells
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Flow cytometry was carried out with 0.5 × 106 sputum cells per tube. Cells were washed with wash buffer. Ice cold 2% Parafix (BD Biosciences, Oxford, UK) was then added and incubated at 4 °C for 10 min. Cells were then washed again. Perm Buffer I (BD Biosciences, Oxford, UK) was then added and incubated for 20 min at 4 °C, followed by a wash in Perm Buffer I. The following antibodies: haptoglobin (antibodies-online, ABIN952678 (primary), APC Conjugation Kit (secondary), Abcam, ab201807), CD163 (Thermo Fisher Scientific, Loughborough, UK 12-1639-42), CD206 (Biolegend, London, UK 321120) and CD45 (Biolegend, London, UK, 304016), were then added and incubated for 45 min at 4 °C. Cells were then washed twice in Perm Buffer I and then resuspended in wash buffer and acquired on FACS Canto II using FACS DIVA software. Data were then analysed using Flowjo software (Treestar, Ashland, OR, USA).
10
Immunohistochemical Profiling of Tumor Samples
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Immmunohistochemistry (IHC) staining was performed using the automated Benchmark platform (Ventana Medical Systems, Tucson, USA), according to the manufacturer's instructions. Four-micrometer-thick TMA sections were mounted onto slides and deparaffinized followed by antigen retrieval using cell conditioning solution and stained with the UltraView™ Universal DAB detection kit (Ventana Medical Systems). The following primary antibodies were used in this study: estrogen receptor (ER; pre-diluted; Ventana Medical Systems), progesterone receptor (PR; pre-diluted; Ventana Medical Systems), human epidermal growth factor receptor 2 (HER2; pre-diluted; Ventana Medical Systems), Ki-67 (1:200; DAKO Co., Carpinteria, USA), CD68 (1:400; Santa Cruz Biotechnology, Dallas, USA), CD11c (1:100; Abcam, Cambridge, USA), and CD163 (1:200; Thermo Fisher Scientific, Waltham, USA).
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