selection classical monocyte isolation kit  (Miltenyi Biotec)

Journal: mAbs

Article Title: Higher order receptor clustering due to the IgG3 subclass is necessary for TLR4 signaling and tolerance induction by novel human anti-TLR4 antibodies

doi: 10.1080/19420862.2025.2515415

Figure Lengend Snippet: IgG3 TV-7D11 induces tolerance in primary human myeloid cells. Human CD14+ monocytes were purified by magnetic bead negative selection. 1 × 10^6 cells/well were seeded in 24-well plates and stimulated with IgG3 8H1 (10 μg/ml), IgG3 TV-7D11 (10 μg/ml), or LPS (.01 μg/ml). After 24 h supernatants were removed and tested for TNF-α by ELISA (a), followed by addition of fresh media and culture for 5 d to allow cells to return to a resting state (b). On day 6, cells were split into 96-well plates at 50,000 cells/well and allowed to rest for 24 h prior to re-challenge with LPS at .01 μg/ml. Supernatants were again collected at 24 h and assayed for TNF-α production by ELISA (c). Data points represent three individuals from two independent experiments. Statistical analysis by ANOVA * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Human CD14+ monocytes were purified by magnetic bead negative selection (Miltenyi, #130–117–337).

Techniques: Purification, Selection, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Pancreatic Adenocarcinoma Up-Regulated Factor (PAUF) Transforms Human Monocytes into Alternative M2 Macrophages with Immunosuppressive Action

doi: 10.3390/ijms252111545

Figure Lengend Snippet: The purity of isolated human peripheral blood CD14 + CD16 − monocytes—Purity investigation of magnetically isolated CD14 + monocytes by flow cytometry and microscope. Data were analyzed using NovoExpress software version 1.5.0. ( A ) The dot plots of forward scatter against side scatter. Representative dot plot of human PBMCs, CD14 − , and CD14 + cells. CD14 + cells were surface labeled with CD14-FITC, CD11c-PE, CD3-APC antibodies. ( B ) Images representative of the subject were taken using a microscope. Monocytes were stained with Wright-Giemsa, revealing dark purple nuclei and a sky blue cytoplasm. Scale bar is 10 μm. 1000× magnification.

Article Snippet: Monocytes were isolated by cluster of differentiation 14 (CD14) negative selection with the human Classical Monocyte Isolation Kit, (#130-117-337, Miltenyi Biotec, Bergisch Gladbach, Germany) using the manual method or the automated machine, an auto magnetic cell separations (autoMACS) Pro instrument (Miltenyi Biotec, Bergisch Gladbach, Germany) as described below.

Techniques: Isolation, Flow Cytometry, Microscopy, Software, Labeling, Staining

Journal: International Journal of Molecular Sciences

Article Title: Pancreatic Adenocarcinoma Up-Regulated Factor (PAUF) Transforms Human Monocytes into Alternative M2 Macrophages with Immunosuppressive Action

doi: 10.3390/ijms252111545

Figure Lengend Snippet: PAUF induces the chemotactic migration of monocytes—Chemotaxis of monocytes by transwell migration assay. ( A ) Schematic of the transwell chemotaxis model. Monocytes were seeded in the upper chamber at 3 × 10 5 cells/200 μL. The chemoattractant PAUF (0.1 μg/mL or 0.5 μg/mL) was added to the lower chamber. PBS served as a control. ( B ) After 20.5 h of incubation, the migrating monocytes were stained with Giemsa stain, and the cells in the fields were counted. ( B` ) The bar chart displayed the percentage of migrated cells compared to the PBS group. ( C ) Human CD14 + monocytes were seeded in the upper chamber at 1.5 × 10 5 cells/200 μL with or without TLR2 inhibitor (125 μM). The lower chamber was supplemented with the chemoattractant PAUF (0.5 μg/mL), Pam 3Cys (100 ng/mL). After 22.5 h of incubation, Giemsa stain was used to stain the migrated monocytes and the cells were counted in captured fields. Representative images of PBS and PAUF treated groups. All scale bars are 200 μm. 100× magnification. ( C` ) Bar chart showing the number of migrated cells, represented as a relative percentage to PBS group. The data were presented as the mean ± standard deviation from a minimum of three independent experiments. *, p < 0.05; ****, p < 0.0001.

Article Snippet: Monocytes were isolated by cluster of differentiation 14 (CD14) negative selection with the human Classical Monocyte Isolation Kit, (#130-117-337, Miltenyi Biotec, Bergisch Gladbach, Germany) using the manual method or the automated machine, an auto magnetic cell separations (autoMACS) Pro instrument (Miltenyi Biotec, Bergisch Gladbach, Germany) as described below.

Techniques: Migration, Chemotaxis Assay, Transwell Migration Assay, Control, Incubation, Staining, Giemsa Stain, Standard Deviation

Journal: bioRxiv

Article Title: High-motility pro-tumorigenic monocytes drive macrophage enrichment in the tumor microenvironment

doi: 10.1101/2024.07.16.603739

Figure Lengend Snippet: ( A ) Illustration cartoon of our in vitro 3D random migration assay. Primary human monocytes isolated from donor PBMCs via negative selection and differentiated M0 (with M-CSF) / M1 (with LPS/IFN- γ)/ M2 (with IL-4/IL-13) and tumor-associated macrophages (with tumor conditioned medium) were embedded into a 1 mg/mL 3D collagen gel and maintained at 37°C, 5% CO2. Overnight tracking videos were recorded, and individual cells were tracked and analyzed with a customized code for trajectories, mean squared displacement (MSD) and other migration parameters. ( B ) Trajectories of M0/M1/M2 and tumor-associated macrophages during 10 h tracking with a time interval of 5 min. The trajectories of all tracked cells were plotted with the same origin. N= 2 donors, n=50 for a total of 100 cells tracked for each phenotype of macrophages. Scale bar, 150μm. ( C ) Mean MSD of all tracked cells plotted against time lag (min) for each phenotype of macrophages. A smaller slope represents smaller displacements within the same migration time, suggesting lower random migration capability. ( D ) Time-independent speeds during the 10 h tracking were calculated via a customized code and plotted. Each dot represents the speed of an individual cell. ( E ) Representative DIC images obtained with a 20x objective for MDA-MB-231 triple negative breast cancer cell (mesenchymal), naïve M0 macrophage (mesenchymal) and monocyte (ameboid) at 0 min, 5 min and 10 min. Morphological features during active cell migration, including dendritic protrusions and blebs, were marked. Scale bar, 20 μm. ( F-H ) Trajectories, speeds and MSDs of MDA-MB-231 cells, M0 macrophages and monocytes during 6 h tracking (3-min time interval). Monocytes and macrophages shared the same donor. More than 100 individual cells were tacked for each cell type. Scale bar, 250μm. ( I-K ) Trajectories, speeds and MSDs of monocytes and naïve M0 macrophages upon exposure to MDA-MB-231 cancer cell conditioned medium. N= 2 donors, n=50 for a total of 100 cells tracked. Scale bar, 250 μm. ( L ) FFPE sections of 72 h spheroids stained for H&E, DAPI (blue channel) and anti-human CD11b (green channel). Representative scanned images were shown for 1 spheroid for illustration purposes. The section of spheroid was divided into tumor core region, invasive margin region and surrounding matrix region based as annotated. ( M ) Unbiased GO enrichment assay on upregulated genes in MDA CM treated monocytes. Top 10 gene sets with highest gene ratios were plotted and cell migration-related biological processes were labeled red. RNA was extracted from monocytes digested from 3D collagen matrix after 24 h treatment with MDA CM. N=3 biological replicates. For migration assays, characterized speeds, MSDs and motile fractions are plotted as mean + standard deviation. Non-parametric Mann-Whitney test was used for statistical analysis comparing 2 groups (****P≤0.0001, ***P≤0.001, ns P˃0.05). Kruskal-Wallis ANOVA with Dunn’s test was used for statistical analysis for multiple comparisons (****P≤0.0001, ***P≤ 0.001).

Article Snippet: Primary human monocytes were isolated from PBMCs using negative selection human classical monocyte isolation kit (Miltenyi Biotec, 130-117-337) according to the manufacturer’s protocol.

Techniques: In Vitro, Migration, Isolation, Selection, Staining, Labeling, Standard Deviation, MANN-WHITNEY

Journal: bioRxiv

Article Title: High-motility pro-tumorigenic monocytes drive macrophage enrichment in the tumor microenvironment

doi: 10.1101/2024.07.16.603739

Figure Lengend Snippet: ( A ) Illustration cartoon of exogeneous cytokine treatment. Monocytes in 3D collagen matrix were incubated for 6 h with 1 ng/mL of IL-6 or IL-8 and then tracked for motility. ( B ) Speeds and motile fractions of monocytes treated with IL-6, IL-8 or IL-6+IL-8. MDA CM- induced TAMo was used as a positive control. ( C ) Surface expressions of IL-6R, IL-8R and CCR2 on classical human monocytes via flow cytometry. Positivity was gated using fluorescence minus one (FMO) control. ( D ) Classical (71.0%), intermediate (11.0%), and non-classical (10.6%) monocytes were gated via CD14/CD16 surface expressions after pan-monocyte isolation. ( E ) Single-cell atlas of human breast cancer with annotated cell subsets (GSE176078) and IL6R and CCR2 expression (high – blue; low – gray). ( F ) Comparison of IL-6 secretion between high-density MDA-MB-231 tumor cores (to mimic tumor microenvironment) and low-density monocytes or MDA-MB-231 cells embedded in 3D collagen gel at 100 cells/mm 3 . N = 3 biological replicates. Unpaired t test with Welch’s correction was used for statistical analysis (**P≤0.01, *P≤0.05, ns P˃0.05). ( G ) IL-6 ELISA from TAMos under indicated conditions. N=3 biological replicates, n = 3 technical replicates. ( H ) Illustration cartoon of functional IL-6R blockade via tocilizumab. 1 ng/mL exogenous IL-6 and 150 nM tocilizumab were added simultaneously to monocytes and incubated for 6 h at 37°C, 5% CO2 before tracking. ( I ) Speeds and motile fractions of monocytes under indicated treatments. ( J ) Top 10 downregulated gene sets in TAMos when IL-6/IL-6R interaction was inhibited. Cell migration related biological processes were labeled red. ( K ) Representative lattice light sheet microscopy images recording active dynamics of cell dendritic protrusions. Scale bar, 5 μm. ( L ) Illustration cartoon showing that IL-6 signaling pathway was inhibited via targeting at downstream transcription factor STAT3 and actin nucleator Arp2/3 complex. ( M-N ) Speeds and motile fractions of monocytes following treatment of 10 μM STAT3 inhibitor S3I-201 or 50 μM Arp2/3 inhibitor CK-666. ( O-P ) Representative western blot results for Arp2 and pMLC2 expression in monocytes treated as indicated. ( Q ) Speeds and motile fractions of TAMos with or without inhibiting myosin II activity using 50 μM blebbistatin. For migration assays, N = 2 donors, n = 50 for a total of 100 individual cells were tracked for each condition in all experiments. Speeds and motile fractions (N = 2 donors, n = 5 technical replicates) are plotted as mean + standard deviation. Non-parametric Mann-Whitney test was used for statistical analysis comparing 2 groups (****P≤0.0001, **P≤0.01, ns P˃0.05). Kruskal-Wallis ANOVA with Dunn’s test was used for statistical analysis comparing groups with control (****P≤0.0001, **P≤0.001, *P≤0.05, ns P˃0.05).

Article Snippet: Primary human monocytes were isolated from PBMCs using negative selection human classical monocyte isolation kit (Miltenyi Biotec, 130-117-337) according to the manufacturer’s protocol.

Techniques: Incubation, Positive Control, Flow Cytometry, Fluorescence, Control, Isolation, Expressing, Comparison, Enzyme-linked Immunosorbent Assay, Functional Assay, Migration, Labeling, Microscopy, Western Blot, Activity Assay, Standard Deviation, MANN-WHITNEY