Journal: Poultry Science

Article Title: Research note: Turkey ( Meleagris gallopavo ) semen CD14 + macrophages – management, key features, and classification – a pilot study with optimized protocols ☆

doi: 10.1016/j.psj.2025.105397

Figure Lengend Snippet: The visualization of turkey macrophages using immunofluorescence (IF) staining. A. A representative turkey semen CD14 + type 1 macrophages (CD14 + iNOS + , akin CD14 + Mф1); B. A representative turkey semen CD14 + type 2 macrophages (CD14 + IL10 + , akin CD14 + Mф2); C. A representative small mononucleated semen CD14 + Mф with an absence of vacuoles; d-F. Hypothetical process of multinucleated giant cell formation from two mononucleated small semen CD14 + Mф; G. Multinucleated giant semen CD14 + Mф with internalized sperm cells. Full arrows indicate vacuoles, and empty arrows indicate selected sperm. Images were captured using a confocal Laser Scanning Microscope (LSM800; Carl-Zeiss, Germany) equipped with an Airyscan super-resolution module (40 × /1.2 NA W c-apochromat) supported by Zen Blue Pro 2.6 software (Carl-Zeiss; Germany).

Article Snippet: Next, cells were triple-washed with PBS and covered with primary mouse anti-CD14 antibodies (orb434888, Biorbyt, United Kingdom, 1:50 in PBS).

Techniques: Immunofluorescence, Staining, Laser-Scanning Microscopy, Software

Journal: Poultry Science

Article Title: Research note: Turkey ( Meleagris gallopavo ) semen CD14 + macrophages – management, key features, and classification – a pilot study with optimized protocols ☆

doi: 10.1016/j.psj.2025.105397

Figure Lengend Snippet: A. Turkey semen CD14 + Mф measured by flow cytometry I-III: The representative cytogrammes show the gating strategy for identifying the different CD14 + Mф subpopulations. B. I-X: Consecutive photographs (10-min intervals) of turkey semen Mф phagocyting zymosan particle imaged using fluorescence microscopy with an Axio Observer. Z1/7 fluorescence microscope (Carl Zeiss, Inc., Oberkochen, Germany) equipped with ZEN 2.3 blue edition software (Carl Zeiss). C. Turkey semen Mф 24 h after seeding (magnification 200 ×), and D. after culture washing (magnification 400 ×). The high incidence of sperm in the cell pool decreases when cultured in high-glucose DMEM and after cell washing. Full arrows indicate selected Mф and empty arrows indicate sperm.

Article Snippet: Next, cells were triple-washed with PBS and covered with primary mouse anti-CD14 antibodies (orb434888, Biorbyt, United Kingdom, 1:50 in PBS).

Techniques: Flow Cytometry, Fluorescence, Microscopy, Software, Cell Culture

Journal: Biochimica et Biophysica Acta. Molecular Basis of Disease

Article Title: Single cell RNA-seq resolution revealed CCR1 + /SELL + /XAF + CD14 monocytes mediated vascular endothelial cell injuries in Kawasaki disease and COVID-19

doi: 10.1016/j.bbadis.2023.166707

Figure Lengend Snippet: The differential activation of CD14 and CD16 monocytes responding to endothelial and epithelial dysfunction. The profile scores of molecules mediating the interplays of classic monocytes with vascular endothelial cells (A-B) and non-classic monocytes with alveolar epithelial cells (C—D). E. Immunostaining for CDH5 and γH2AX in coronary artery cryo-section between KD patient and healthy donor, indicating endothelial injuries in KD. F. Expression level of CCR1 , DYSF , SELL , LMNB1, and XAF1 in CD14 classical monocytes among five groups. G. UMAP projection of CCR1 , DYSF , SELL , LMNB1, and XAF1 positive cells, respectively . H. UMAP projection of CCR1 , SELL and XAF1 triple positive monocytes among five groups. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, Wilcoxon rank-sum test, adjusted for Bonferroni post hoc test, had been applied.

Article Snippet: Cell smears fixated in 4 % PFA were blocked in blocking buffer (PBS containing 1 % BSA, 0.1 % Triton X-100, 5 % goat serum) for 1 h at room temperature, followed by incubation with rabbit anti-Dysferlin (1:200, Abcam, cat # ab124684), rabbit anti-XAF1 (1:200, Abcam cat #ab2254) antibodies and mouse anti-CD14 (1:200, Abcam cat #ab181470) diluted in PBS containing 1 % BSA, 5 % goat serum at 4 °C overnight.

Techniques: Activation Assay, Immunostaining, Expressing

Journal: Biochimica et Biophysica Acta. Molecular Basis of Disease

Article Title: Single cell RNA-seq resolution revealed CCR1 + /SELL + /XAF + CD14 monocytes mediated vascular endothelial cell injuries in Kawasaki disease and COVID-19

doi: 10.1016/j.bbadis.2023.166707

Figure Lengend Snippet: SELL+/CCR1+/XAF1+ CD14 monocytes enhanced the adhesion and damages to endothelial cells. A. Flow cytometry identified the higher percentages and median fluorescence intensity of SELL, CCR1, and LMNB1 in KD and COV. B. Immunostaining for CD14 and DYSF, XAF1 in isolated PBMCs. C. The ratio of DYSF positive CD14 monocytes in total classic monocytes among KD, COV, FLU, and healthy donors. And the median fluorescence intensity of XAF1 in CD14 monocytes among KD, FLU, and healthy donors. D-E. THP-1 had been stained with Calcein AM and transfected siRNAs of CCR1 , DYSF , SELL , LMNB1 , and XAF1 before co-culture with HUVECs. Then the numbers of adhesion THP-1 with HUVECs were counted by every 20× field view. F. The expressions of TNFa and IL6 in HUVECs, and the ratio of γH2AX + cells in HUVECs after co-cultured with THP-1, which had been transfected with siRNAs of CCR1 , DYSF , SELL , LMNB1 , and XAF1 . G. The inhibition of SELL , CCR1 and XAF1 together in THP-1 significantly reduced the adhesion between monocytes and endothelial cells. H. Collaborated inhibiton of SELL , CCR1 and XAF1 in THP-1 decreased the expression of IL6 and TNF-α in HUVECs with lower ratio of rH2AX+ cells after co-culture with THP-1. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, Two-way analysis of variance with Bonferroni post hoc test was performed to analyze data. Bar, 100 μm.

Article Snippet: Cell smears fixated in 4 % PFA were blocked in blocking buffer (PBS containing 1 % BSA, 0.1 % Triton X-100, 5 % goat serum) for 1 h at room temperature, followed by incubation with rabbit anti-Dysferlin (1:200, Abcam, cat # ab124684), rabbit anti-XAF1 (1:200, Abcam cat #ab2254) antibodies and mouse anti-CD14 (1:200, Abcam cat #ab181470) diluted in PBS containing 1 % BSA, 5 % goat serum at 4 °C overnight.

Techniques: Flow Cytometry, Fluorescence, Immunostaining, Isolation, Staining, Transfection, Co-Culture Assay, Cell Culture, Inhibition, Expressing

Journal: Frontiers in Physiology

Article Title: Using Imaging Mass Cytometry to Define Cell Identities and Interactions in Human Tissues

doi: 10.3389/fphys.2021.817181

Figure Lengend Snippet: List of antibodies used on human kidney tissue.

Article Snippet: CD14 , Monocytes/Macrophages , Rabbit , EPR3653 , 144Nd , 1:50 , Fluidigm , .

Techniques: Membrane

Journal: Frontiers in Immunology

Article Title: PFKFB3 overexpression in monocytes of patients with colon but not rectal cancer programs pro-tumor macrophages and is indicative for higher risk of tumor relapse

doi: 10.3389/fimmu.2022.1080501

Figure Lengend Snippet: Monocyte subpopulations` content in healthy individuals and CRC patients.

Article Snippet: For immunofluorescence (IF) staining, tumor FFPE clinical samples were treated with xylol solution and blocked with 3% BSA in PBS for 45 min, incubated with a combination of primary antibodies for 1,5 h; washed, and incubated with a combination of appropriate secondary antibodies for 45 min. Anti-PFKFB3 rabbit monоclonal antibody (1:50, #ab181861, Abcam, USA); anti-CD68 monoclonal mouse antibody (1:100, #NBP2-44539, clone KP1, Novus Biologicals); anti-CD14 polyclonal sheep antibody (1:50, #BAF383, R&D Systems) were used.

Techniques:

Journal: Frontiers in Immunology

Article Title: PFKFB3 overexpression in monocytes of patients with colon but not rectal cancer programs pro-tumor macrophages and is indicative for higher risk of tumor relapse

doi: 10.3389/fimmu.2022.1080501

Figure Lengend Snippet: The distribution of CD163+ and CCR2+ peripheral blood monocytes in patients with colon and rectal cancers. Individual profiles of CCR2+ and CD163+ monocyte subsets for each patient with rectal and colon cancers. (A) , The distribution of monocytes of classical (CD14+CD16-), intermediate (CD14+CD16+) and non-classical (CD14-CD16+) populations expressing CCR2 (upper panel) and CD163 (lower panel) is demonstrated before and after NAC and after surgical resection in rectal cancer patients. (B) , The distribution of monocytes of classical (CD14+CD16-), intermediate (CD14+CD16+) and non-classical (CD14-CD16+) populations expressing CCR2 (upper panel) and CD163 (lower panel) is demonstrated before and after surgical resection in colon cancer patients. (C) , Associations of CCR2-expressing monocyte subsets with hematogenous and lymphatic metastasis in rectal cancer patients. (D) , Associations of CD163-expressing monocyte subsets with hematogenous and lymphatic metastasis in colon cancer patients. M 0 , metastasis-negative status, M 1 , metastasis-positive status. N 0 , lymph node-negative status, N 1-3 , lymph node-positive status.

Article Snippet: For immunofluorescence (IF) staining, tumor FFPE clinical samples were treated with xylol solution and blocked with 3% BSA in PBS for 45 min, incubated with a combination of primary antibodies for 1,5 h; washed, and incubated with a combination of appropriate secondary antibodies for 45 min. Anti-PFKFB3 rabbit monоclonal antibody (1:50, #ab181861, Abcam, USA); anti-CD68 monoclonal mouse antibody (1:100, #NBP2-44539, clone KP1, Novus Biologicals); anti-CD14 polyclonal sheep antibody (1:50, #BAF383, R&D Systems) were used.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: PFKFB3 overexpression in monocytes of patients with colon but not rectal cancer programs pro-tumor macrophages and is indicative for higher risk of tumor relapse

doi: 10.3389/fimmu.2022.1080501

Figure Lengend Snippet: An activator of glycolysis PFKFB3 is overexpressed in colon cancer and is indicative for higher risk of tumor relapse in colon cancer but not rectal cancer. (A) , Сolon cancer tissue is massively infiltrated by PFKFB3-positive monocytes. IF/confocal microscopy analysis was performed for 10 colon tumor tissues. The infiltration of CD14+CD68+PFKFB3+ cells was found in all samples. Representative image is given from one patient. Scale bar corresponds to 50 µm in main image and 20 µm in zoom image. (B) , Spearman correlation coefficients between PFKFB3 expression, M2 macrophage gene expressions and predicted cell abundance scores, FDR<0.05. (C) , Predicted cell composition of CD45+ AOIs and hierarchical clustering of AOIs. (D) , Difference in monocyte and macrophage cell abundance scores between the CD45+ AOIs in colon and rectal cancers (the Mann-Whitney U test was applied). (E) , PFKFB3 gene expression is elevated in patients with recurrence and larger tumor size in colon cancer. Variance in PFKFB3 expression was stabilized via the variance stabilizing transformation (VST). (F) , PFKFB3 had prognostic significance for the DFS and OS. High-risk group had worse survival rates compared to low-risk group. ROC analysis and Kaplan–Meier method were applied.

Article Snippet: For immunofluorescence (IF) staining, tumor FFPE clinical samples were treated with xylol solution and blocked with 3% BSA in PBS for 45 min, incubated with a combination of primary antibodies for 1,5 h; washed, and incubated with a combination of appropriate secondary antibodies for 45 min. Anti-PFKFB3 rabbit monоclonal antibody (1:50, #ab181861, Abcam, USA); anti-CD68 monoclonal mouse antibody (1:100, #NBP2-44539, clone KP1, Novus Biologicals); anti-CD14 polyclonal sheep antibody (1:50, #BAF383, R&D Systems) were used.

Techniques: Confocal Microscopy, Expressing, MANN-WHITNEY, Gene Expression, Transformation Assay