mouse anti cd68 plus rabbit anti cd163  (Proteintech)

Journal: Bioactive Materials

Article Title: Immune regulative GelMA&Zn 2+ /Ce 3+ -whitlockite scaffolds with continuous ions release for bone regeneration

doi: 10.1016/j.bioactmat.2025.11.009

Figure Lengend Snippet: Evaluation of immune inflammation (RAW264.7) in the scaffolds. A, B) Immunofluorescent staining results and quantitative fluorescence intensity of CD206 in RAW264.7 cells co-cultured with different sample extracts, with CD206 (M2 marker) shown in red (scale bar = 50 μm). C, D) Immunofluorescent staining results and quantitative fluorescence intensity of CD86 in RAW264.7 cells co-cultured with different sample extracts, with CD86 (M1 marker) shown in green (scale bar = 50 μm). E, F) Immunofluorescent staining results and quantitative fluorescence intensity of CD163 in RAW264.7 cells co-cultured with different sample extracts, with CD163 (M2 marker) shown in green (scale bar = 50 μm). G) Flow cytometry analysis of CD86 and CD11b positive cells in inflammatory RAW264.7 cells. H) Fluorescence enzyme labeling for the quantitative analysis of ROS content in macrophages. I) Cell viability of RAW264.7 cells co-cultured with different sample extracts for 1, 3, and 5 days. J) NO content in RAW264.7 cells co-cultured with different sample extracts. K) DCFH-DA fluorescent probe detection to evaluate ROS content in macrophages after stimulation with different sample extracts (scale bar = 100 μm). P values were determined using one-way ANOVA (∗∗∗∗P < 0.0001, ∗∗∗P < 0.001, ∗∗P < 0.01, ∗P < 0.05, N.S., not significant).

Article Snippet: Following stimulation, the cells were blocked with 5 % bovine serum albumin (BSA) for 2 h at room temperature, washed three times with PBS, and incubated overnight at 4 °C with a CD86 polyclonal antibody (1:600, 13395-1-AP, Proteintech, USA) and CD163 polyclonal antibody (1:600, 16646-1-AP, Proteintech, USA).

Techniques: Staining, Fluorescence, Cell Culture, Marker, Flow Cytometry, Labeling

Journal: Bioactive Materials

Article Title: Immune regulative GelMA&Zn 2+ /Ce 3+ -whitlockite scaffolds with continuous ions release for bone regeneration

doi: 10.1016/j.bioactmat.2025.11.009

Figure Lengend Snippet: Evaluation of immune inflammation (BMDM) in the scaffolds. A, B) Immunofluorescent staining results and quantitative fluorescence intensity of CD86 in BMDM cells co-cultured with different sample extracts, with CD86 (M1 marker) shown in green (scale bar = 50 μm). C, D) Immunofluorescent staining results and quantitative fluorescence intensity of CD163 in BMDM cells co-cultured with different sample extracts, with CD163 (M2 marker) shown in green (scale bar = 50 μm). P values were determined using one-way ANOVA (∗∗∗∗P < 0.0001, ∗∗∗P < 0.001, ∗∗P < 0.01, ∗P < 0.05, N.S., not significant).

Article Snippet: Following stimulation, the cells were blocked with 5 % bovine serum albumin (BSA) for 2 h at room temperature, washed three times with PBS, and incubated overnight at 4 °C with a CD86 polyclonal antibody (1:600, 13395-1-AP, Proteintech, USA) and CD163 polyclonal antibody (1:600, 16646-1-AP, Proteintech, USA).

Techniques: Staining, Fluorescence, Cell Culture, Marker

Journal: bioRxiv

Article Title: YAP/TEAD drives treatment-induced adaptive immunosuppression in EGFR-mutant lung cancer

doi: 10.64898/2026.01.22.701073

Figure Lengend Snippet: A) Experimental set-up to generate DTP cells, collection of CM, and exposure to monocytes and macrophages. B) HCC827 cells expressing pLEX307-FUCCI cell cycle reporter were treated with either DMSO, 100 nM osimertinib alone, or in combination with 30 nM trametinib for 10 days. C) THP-1 cells were treated as in (B), and the expression of M2 marker genes (CD163 and MRC1/CD206) was analyzed with RT-qPCR. D) CM was collected as in (B), with the addition of either short (48 h) or long (13 days) drug holidays, followed by THP-1 incubation as in (C). E) CD14⁺ monocyte chemotaxis toward CM from DMSO- or osimertinib/trametinib–treated cancer cells. Chemotaxis was measured using Incucyte live-cell imaging. Data shown as a percentage of cells migrated after 24 hours. F) RNA-sequencing of monocyte-derived macrophages treated according to (A), with M1 and M2 gene set expression scored relative to expression of all genes. G) GSEA of REACTOME pathways in monocyte-derived macrophages treated as in (F). H) Relative expression of genes driving the enrichment of binding and uptake of ligands by scavenger receptors -gene set (G). I) Flow cytometry analysis of cell surface expression of CD163 in monocytes and monocyte-derived macrophages (J) exposed to DTP CM. DTP = drug-tolerant persister cells, achieved with osimertinib/trametinib treatment for 12 days, CM = conditioned medium, GSEA = gene set enrichment analysis. Data shown as mean ± SD in C and D. One-way ANOVA (C, D) or paired t-test (E, F, I, J) was used to assess statistical significance. ***, P-value <0.001; **, P-value <0.01; *, P-value < 0.05

Article Snippet: The following reagents were used to determine gene expression levels; Taqman 2X Universal master mix (cat# 4440047), and 20X gene expression assays GAPDH (Hs02758991_g1), ACTB (Hs99999903_m1), TBP (Hs00427620_m1), MRC1/CD206 (Hs00267207_m1), and CD163 (Hs00174705_m1).

Techniques: Expressing, Marker, Quantitative RT-PCR, Incubation, Chemotaxis Assay, Live Cell Imaging, RNA Sequencing, Derivative Assay, Binding Assay, Flow Cytometry

Journal: bioRxiv

Article Title: YAP/TEAD drives treatment-induced adaptive immunosuppression in EGFR-mutant lung cancer

doi: 10.64898/2026.01.22.701073

Figure Lengend Snippet: A) Experimental set-up for conditioned media collection and YAP/TEAD inhibition in DTP cells. B) RNA-sequencing analysis of the expression of genes encoding for secreted proteins in HCC827 and HCC4006 cell lines treated as in (A). C) YAP-driven secretome in HCC827 and HCC4006 cells, determined by genes upregulated in DTPs, and downregulated upon TEAD inhibition. D) Shared YAP-driven secretome in HCC827 and HCC4006. E) ChIP-seq analysis of YAP binding of shared secretome genes. F) List of shared YAP-driven secretome from (D), with genes previously associated with macrophage activation, migration, or polarization highlighted in bold. G) CD163 expression of THP-1 cells treated with HCC827 conditioned media collected as in (A) using 500 nM ORM-47286 or 10 mM MYF-01-37 for TEAD inhibition. H) Flow cytometry analysis of CD163 and TREM2 cell surface expression in human peripheral blood monocytes exposed to HCC827 conditioned media. Data in (G) presented as mean ± SD. Unpaired (G) and paired (H) one-way ANOVA was used to assess statistical significance. **, P-value <0.01; *, P-value < 0.05.

Article Snippet: The following reagents were used to determine gene expression levels; Taqman 2X Universal master mix (cat# 4440047), and 20X gene expression assays GAPDH (Hs02758991_g1), ACTB (Hs99999903_m1), TBP (Hs00427620_m1), MRC1/CD206 (Hs00267207_m1), and CD163 (Hs00174705_m1).

Techniques: Inhibition, RNA Sequencing, Expressing, ChIP-sequencing, Binding Assay, Activation Assay, Migration, Flow Cytometry

Journal: bioRxiv

Article Title: Mechanobiological Specialization of Choroid Plexus Macrophages Defined by Titin Expression

doi: 10.64898/2026.01.20.700716

Figure Lengend Snippet: Titin-expressing macrophages are present in the choroid plexus. (A) Heatmap of the top eight differentially expressed genes in each of the three ChP macrophage sub-clusters. Gene expression levels are represented on a normalized gradient scale. (B) DAB immunohistochemical stain of a 5 µm FFPE section of control ChP tissue using a CD163 antibody. CD163-positive macrophages are marked by brown staining, while cell nuclei are counterstained blue with hematoxylin, indicating their distribution within the vascularized ChP tissue. Scale bar = 40 µm. (C) Violin plots illustrating TTN expression levels in ChP samples from Alzheimer’s disease and control conditions. Each plot shows the distribution, density, and variability of TTN transcript counts in ChP macrophages within each condition. (D) Schematic representation of the experimental workflow for evaluation of TTN RNA enrichment in macrophages, involving tissue dissociation, CD163 positive cell enrichment, RNA isolation, cDNA synthesis, PCR amplification using isoform-specific TTN primers, and ΔΔCq calculation with GAPDH used for normalization. (E) Fold changes, computed from ΔΔCq, in TTN isoform expression relative to GAPDH in CD163-bead enriched macrophages relative to whole tissue. The x-axis represents different primer sets (1, 3, 7, and 12), and the y-axis shows fold changes (log scale). Each point represents a single donor sample/primer set pair. (F) Schematic representation of the TTN gene with highlighted primer binding sites, with genomic coordinates on chromosome 2, indicating selected splicing sites and isoforms of TTN. (G) Immunofluorescence images of ChP with anti-CD68 (red) and anti-titin (green) primary antibodies, co-stained with DAPI (blue), showing cytoplasmic titin protein in ChP macrophage cytoplasm. Merged images are in right column. Scale bars = 20 µm.

Article Snippet: RNA was extracted from CD163 microbead-separated macrophages (Miltenyi Biotec, Cat. #130-124-420) using the RNeasy Mini Kit (Qiagen), following the manufacturer’s protocol.

Techniques: Expressing, Gene Expression, Immunohistochemical staining, Staining, Control, Isolation, cDNA Synthesis, Amplification, Binding Assay, Immunofluorescence