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: 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: Acta Pharmaceutica Sinica. B

Article Title: PPAR α affects hepatic lipid homeostasis by perturbing necroptosis signals in the intestinal epithelium

doi: 10.1016/j.apsb.2024.08.021

Figure Lengend Snippet: Pparα deficiency in intestinal epithelium promotes hepatic steatosis and fibrosis in mice fed a high-fat plus high-sucrose diet (HFHS). (A) Schematic representation of HFHS experimental design. (B) Representative images of liver tissue stained with H&E and Oil Red O after short-term HFHS treatment ( n = 5). (C) Relative mRNA levels of Col1a1 , β-Pdgfr , Tgf-β , and Timp-1 in the liver after short-term HFHS treatment ( n = 5). (D) Relative mRNA levels of F4/80 , Clec4f , and Cd14 in the liver after short-term HFHS treatment ( n = 5). (E) Body weight of mice after long-term HFHS treatment ( n = 15). (F) Liver weight of mice after long-term HFHS treatment ( n = 15). (G) The length of the small intestine after long-term HFHS treatment ( n = 15). (H) Serum endotoxin levels after long-term HFHS treatment ( n = 15). (I) The endotoxin levels in the liver after long-term HFHS treatment ( n = 15). (J) Relative mRNA levels of F4/80 , Clec4f , and Cd14 in the liver after short-term HFHS treatment ( n = 5). (K) Relative mRNA levels of Col1a1, β-Pdgfr , Tgf-β , and Timp1 in the liver after the HFHS treatment for 16 weeks ( n = 5). (L) Representative images of liver tissue stained with H&E, Oil Red O, and Masson's trichrome and immunohistochemical staining for α -SMA and TGF- β after long-term HFHS treatment ( n = 5). (M) Levels of α -SMA and TGF- β were quantified using Image J software and expressed as AODs. Data are shown as the mean ± SD. For the two groups, statistical significance was tested by unpaired Student's t -test; for more than two groups, statistical significance was tested by one-way ANOVA followed by the least significant difference (LSD) test; ∗ P < 0.05 , ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: The primary antibodies were as follows: polyclonal rabbit anti-mouse F4/80 (1:100, #A1256, ABclonal), monoclonal rabbit anti-mouse CD14 (1:100, #A19011, ABclonal), polyclonal rabbit anti-mouse FADD (1:100, #A20252, ABclonal), monoclonal rabbit anti-mouse p-RIPK3 (1:100, #91702, CST), monoclonal rabbit anti-mouse p-MLKL (1:200, #37333, CST), monoclonal rabbit anti-mouse cleaved-caspase 8 (1:100, #8592, CST), polyclonal rabbit anti-mouse TRIM38 (1:100, #13405-1-AP, Proteintech).

Techniques: Staining, Immunohistochemical staining, Software

Journal: Acta Pharmaceutica Sinica. B

Article Title: PPAR α affects hepatic lipid homeostasis by perturbing necroptosis signals in the intestinal epithelium

doi: 10.1016/j.apsb.2024.08.021

Figure Lengend Snippet: Pparα deficiency in intestinal epithelium activates inflammatory signaling pathways in both the ileum and liver. (A) Endotoxin in serum and liver ( n = 10). (B) Immunofluorescent staining of F4/80 (red) in the liver of 8-week-old mice ( n = 5). (C) Protein levels of F4/80 in the liver of 8-week-old mice ( n = 3). (D) Relative mRNA levels of F4/80 , Clec4f , and Cd14 in the liver of 8-week-old mice ( n = 5). (E) Relative mRNA levels of Il-1β and Tnf-α in the liver of 8-week-old mice ( n = 5). (F) Relative mRNA levels of Lbp in the liver of 8-week-old mice ( n = 5). (G) Representative H&E staining, immunohistochemical staining for AP, and immunofluorescent staining for CD14 (green) in the ileum of 8-week-old mice ( n = 5). (H) Protein levels of CD14 and relative mRNA levels of Lbp and CD14 in the ileum of 8-week-old mice ( n = 5). (I) Enrichment graph of NOD-like receptor signaling dataset performed with ileum samples from 8-week-old Pparα ΔIE mice ( n = 3). (J) Heatmap representation of genes involved in innate antibacterial defense in 8-week-old Pparα ΔIE ileum relative to Pparα fl/fl ( n = 3). (K) Relative mRNA levels of Defa21 , Defa22 , Defa3 , Defa5 , and Reg3β in the ileum from 8-week-old mice ( n = 5). (L) Schematic representation of high-fructose corn syrup (HFCS) experimental design. (M) Representative images stained with H&E and Oil Red O, and immunofluorescent staining for F4/80 (red) in liver tissue from 8-week-old mice exposed to HFCS for 14 days ( n = 5). (N) Representative H&E image and immunohistochemical staining for AP in the ileum of 8-week-old mice exposed to HFCS for 14 days ( n = 5). (O) Serum endotoxin levels in 8-week-old mice exposed to HFCS for 14 days ( n = 8). AP: alkaline phosphatase staining. Data are shown as the mean ± SD. An unpaired two-tailed Student's t -test; ∗ P < 0.05 , ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: The primary antibodies were as follows: polyclonal rabbit anti-mouse F4/80 (1:100, #A1256, ABclonal), monoclonal rabbit anti-mouse CD14 (1:100, #A19011, ABclonal), polyclonal rabbit anti-mouse FADD (1:100, #A20252, ABclonal), monoclonal rabbit anti-mouse p-RIPK3 (1:100, #91702, CST), monoclonal rabbit anti-mouse p-MLKL (1:200, #37333, CST), monoclonal rabbit anti-mouse cleaved-caspase 8 (1:100, #8592, CST), polyclonal rabbit anti-mouse TRIM38 (1:100, #13405-1-AP, Proteintech).

Techniques: Protein-Protein interactions, Staining, Immunohistochemical staining, Two Tailed Test

Journal: Acta Pharmaceutica Sinica. B

Article Title: PPAR α affects hepatic lipid homeostasis by perturbing necroptosis signals in the intestinal epithelium

doi: 10.1016/j.apsb.2024.08.021

Figure Lengend Snippet: Pparα deficiency in intestinal epithelium increases the translocation of gut-derived antigens into the liver. (A) Intestinal permeability assessment (FITC-dextran, 4 kD) in 8-week-old mice ( n = 10). (B) Relative mRNA levels of Zo-1 and Cldn8 in the ileum from 8-week-old mice ( n = 5). (C) The relative proportion of bacterial species in the cecum content by 16S rRNA gene sequencing ( n = 6). (D) Bugbase phenotypic prediction of gut microbiota in 8-week-old mice ( n = 6). (E) The mRNA and protein levels of PV1 in the ileum of 8-week-old mice ( n = 5). (F) Transmission electron microscopy images of the diaphragm (red star) in the capillaries from ileum sections in 24-week-old mice ( n = 3). (G) Representative images of fluorescence microscopy and transmission electron in 8-week-old mice treated with FITC-LPS (green) or Au-LPS ( n = 3–5). (H) Portal HDL-C levels in 8-week-old mice ( n = 10). (I) The protein levels of APOA1 and ABCA1 in the ileum of 8-week-old mice ( n = 5). (J) Relative mRNA levels of Apoa1 , Pon1 , and Pon3 in the ileum of 8-week-old mice ( n = 5). (K) Serum APOA1 levels in 8-, 16- and 32-week-old mice ( n = 8–10). (L) Serum APOA1 levels in 8-week-old mice exposed to HFCS for 14 days ( n = 8). (M) Serum APOA1 levels in 16-week-old Pparα Δhep mice ( n = 10). (N) Schematic representation of a cocktail of broad-spectrum antibiotics (Abx) experimental design. (O) Representative images stained with H&E and Oil Red O, and immunofluorescent staining for F4/80 (red) in the liver from 8-week-old mice treated with Abx ( n = 5). (P) Triglyceride in serum and liver treated with Abx ( n = 10). (Q) Relative mRNA levels of genes related to triglyceride accumulation in the liver from 8-week-old mice treated with Abx ( n = 5). (R) Hepatic levels of cytokines from 8-week-old mice treated with Abx ( n = 5). (S) Protein levels of F4/80 in the liver of 8-week-old mice treated with Abx ( n = 3). (T) Relative mRNA levels of F4/80, Clec4f , and Cd14 in the liver of 8-week-old mice treated with Abx ( n = 5). LD: lipid droplet; M: mitochondria; FITC-LPS: fluorescein isothiocyanate (FITC)-LPS; Au-LPS: LPS-gold-complexes. Data are shown as the mean ± SD. An unpaired two-tailed Student's t -test; ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: The primary antibodies were as follows: polyclonal rabbit anti-mouse F4/80 (1:100, #A1256, ABclonal), monoclonal rabbit anti-mouse CD14 (1:100, #A19011, ABclonal), polyclonal rabbit anti-mouse FADD (1:100, #A20252, ABclonal), monoclonal rabbit anti-mouse p-RIPK3 (1:100, #91702, CST), monoclonal rabbit anti-mouse p-MLKL (1:200, #37333, CST), monoclonal rabbit anti-mouse cleaved-caspase 8 (1:100, #8592, CST), polyclonal rabbit anti-mouse TRIM38 (1:100, #13405-1-AP, Proteintech).

Techniques: Translocation Assay, Derivative Assay, Permeability, Sequencing, Transmission Assay, Electron Microscopy, Fluorescence, Microscopy, Staining, Two Tailed Test

Journal: Acta Pharmaceutica Sinica. B

Article Title: PPAR α affects hepatic lipid homeostasis by perturbing necroptosis signals in the intestinal epithelium

doi: 10.1016/j.apsb.2024.08.021

Figure Lengend Snippet: Butyric acid attenuates PPAR α -induced necroptosis in HIEC-6 cells and ameliorates hepatic steatosis in Pparα ΔIE mice. (A) Relative CREB3L3 mRNA level in HIEC-6 cells transfected with the PPAR α expression vector ( n = 3). (B) Relative mRNA level of apoptotic and necroptosis signaling components in HIEC-6 cells transfected with the CREB3L3 expression vector ( n = 3). (C) Relative mRNA level of apoptotic and necroptosis signaling components in HIEC-6 cells treated with TNF- α ( n = 3). (D) Effects of GW6471 on RIPK3 and MLKL promoter-driven luciferase activities in human intestinal epithelial HIEC-6 cells ( n = 3). (E) Relative CREB3L3 mRNA level in HIEC-6 cells treated with butyric acid ( n = 3). (F) Relative mRNA level of necroptosis signaling components in HIEC-6 cells treated with TNF- α and butyric acid ( n = 3). (G) Schematic representation of the experimental design for butyric acid treatment. H, Relative CREB3L3 mRNA level in Pparα fl/fl and Pparα ΔIE mice treated with butyric acid ( n = 5). (I) Relative mRNA level of necroptosis signaling components in Pparα ΔIE mice treated with butyric acid ( n = 5). (J) Representative immunohistochemical staining for cleaved-caspase 3 and MLKL, and immunofluorescent staining for cleaved-caspase 8 (green), p-RIPK3 (red), and p-MLKL (red) in the ileum after butyric acid treatment ( n = 5). (K) Representative images stained with H&E and Oil Red O, and immunofluorescent staining for F4/80 (red) in liver tissue after butyric acid treatment ( n = 5). (L) Relative mRNA levels of genes related with lipolysis, lipogenesis, and lipid droplet proteins in the liver after butyric acid treatment ( n = 5). (M) Triglyceride in serum and liver after butyric acid treatment ( n = 10). (N) Hepatic levels of cytokines in mice after butyric acid treatment ( n = 5). (O) Protein levels of F4/80 in the liver after butyric acid treatment ( n = 3). (P) Relative mRNA levels of F4/80 , Clec4f , and Cd14 in the liver after butyric acid treatment ( n = 5). Buty: butyric acid. Data are shown as the mean ± SD. For the two groups, statistical significance was tested by unpaired Student's t -test; for more than two groups, statistical significance was tested by one-way ANOVA followed by the least significant difference (LSD) test; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Article Snippet: The primary antibodies were as follows: polyclonal rabbit anti-mouse F4/80 (1:100, #A1256, ABclonal), monoclonal rabbit anti-mouse CD14 (1:100, #A19011, ABclonal), polyclonal rabbit anti-mouse FADD (1:100, #A20252, ABclonal), monoclonal rabbit anti-mouse p-RIPK3 (1:100, #91702, CST), monoclonal rabbit anti-mouse p-MLKL (1:200, #37333, CST), monoclonal rabbit anti-mouse cleaved-caspase 8 (1:100, #8592, CST), polyclonal rabbit anti-mouse TRIM38 (1:100, #13405-1-AP, Proteintech).

Techniques: Transfection, Expressing, Plasmid Preparation, Luciferase, Immunohistochemical staining, Staining

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