Journal: Diabetes & Metabolism Journal

Article Title: Effects of CXCR1/2 Blockade with Ladarixin on Streptozotocin-Induced Type 1 Diabetes Mellitus and Peripheral Neuropathy and Retinopathy in Rat

doi: 10.4093/dmj.2024.0504

Figure Lengend Snippet: Late ladarixin (LDX) normalized signs of streptozotocin (STZ)-induced diabetic retinopathy and diabetic nephropathy: retinal expression of CXC motif chemokine ligand 8 (CXCL8), CXC motif chemokine receptor 1 (CXCR1), citrullinated histone H3 (CitH3) myeloperoxidase (MPO), CD34, and vascular endothelial growth factor (VEGF) and of plasma creatinine levels in STZ-treated rats. Effects of LDX (15 mg/kg, intragastric [i.g.]) treatment (from week 8 to 12) in control (CTRL), STZ/vehicle (VEH), and STZ/LDX groups on the representative images of (A, H) CitH3, (B, I) MPO, (C, J) CXCL8, (D, K) CXCR1, and (E, L) CXCR2 expression in the retina layers and relative quantification measured as fluorescence intensity per area at different time points post-STZ injection. Effects of LDX (15 mg/kg, i.g.) treatment (from week 8 to 12) in CTRL, STZ/VEH, and STZ/LDX groups on the representative images of (F, M) CD34 and (G, N) VEGF expression in the retina layers and relative quantification measured as fluorescence intensity per area at different time points post-STZ injection. Data are represented as mean±standard error of the mean ( n =3). (O) Effects of LDX (15 mg/kg, i.g.) treatment (from week 8 to 12) in CTRL, STZ/VEH, and STZ/LDX groups on creatinine (pg/mL) measured at 9- and 12-week post-STZ injection. Data are represented as mean±standard error of the mean ( n =4). The threshold of 0.05 was used for determining statistical significance. The magnification of immunofluorescence picture is of ×20. GRO/CINC1, growth-regulated gene product/cytokine-induced neutrophil chemoattractant 1. a P <0.0001, b P <0.001, and c P <0.0001 indicate significant differences vs. CTRL and STZ/VEH.

Article Snippet: Likewise, the eyes sections with antibodies against histone H3 (rabbit anti-histone H3 antibody; 1:200; Abcam), MPO (mouse anti-MPO antibody; 1:50; Abcam), CXCL1 (mouse CXCL8 monoclonal antibody; 1:400; Thermo Fisher Scientific, Waltham, MA, USA), CXCR1 (rabbit CXCR1 polyclonal antibody; 1:100; Elabscience, Houston, TX, USA), CXCR2 (rabbit CXCR2 polyclonal antibody; 1:100; Elabscience), VEGF (rabbit VEGF recombinant rabbit monoclonal antibody; 1:200; Thermo Fisher Scientific), and CD34 (rabbit CD34 polyclonal antibody; 1:100; Elabscience).

Techniques: Expressing, Clinical Proteomics, Control, Quantitative Proteomics, Fluorescence, Injection, Immunofluorescence

Journal: EBioMedicine

Article Title: A liquid biopsy for detecting circulating mesothelial precursor cells: A new biomarker for diagnosis and prognosis in mesothelioma

doi: 10.1016/j.ebiom.2020.103031

Figure Lengend Snippet: Overview of mesothelial precursor cells (MPC) analysis using a microfluidic approach. (a) Sample preparation for MPC analysis is quick and simple. MSLN+ cells are tagged with biotinylated anti-MSLN antibody and further conjugated with anti-biotin magnetic nanoparticle (MNP) complex. Magnetically labeled samples are loaded into the microfluidic chip at 500µL/h with external magnets placed above and below the device to facilitate magnetic trapping of targeted cells. (b) Multiple populations of MPC exist in circulation and are subcategorized based on varying combinations of CD34 and/or CD90 in conjunction with mesothelin (MSLN). Image created with BioRender. (c) Overview of MesoFind microfluidic chip for MPC isolation. The microfluidic device contains 8 different zones with a sequential increase in height from 50µm to 400µm (inlet-to-outlet) and features varying average linear flow velocities (1.0x, 0.50x, 0.33x, 0.25x, 0.20x, 0.17x, 0.14x, 0.13x). Cells magnetically labeled with mesothelin are captured in different zones based on the relative amount of magnetic content. Cells with higher MSLN expression (high magnetic content) are captured in earlier zones whereas cells with less MSLN (low magnetic content) are captured in later zones. Captured cells are fluorescently labeled with CD34 and CD90 antibodies to identify and isolate different MPC subpopulations. (d) MSLN expression profiles are generated for each MPC subpopulation based on the captured location on the chip. Each MPC subtypes are predicted to exhibit different clinical characteristics in patients and their expression of MSLN correlates to the severity of the conditions.

Article Snippet: Afterwards, cells were then incubated in the dark for 1 hour at room temperature with a cocktail of antibodies containing either 2mg/mL of anti-CD34 AF488 (bs-8996R-A488, Bioss Antibodies, USA), 2mg/mL of anti-CD90 AF555 (bs-0778R-A555, Bioss Antibodies, USA), and 2 mg/mL streptavidin AF647 (405237, Biolegend, USA) for RN5 samples or 1mg/mL of anti-CD34 AF488 (ab195013, Abcam, Canada), 0.5mg/mL of anti-CD90 AF555 (bs-10430R-A555, Bioss Antibodies, USA), and 2 mg/mL streptavidin AF647 (405237, Biolegend, USA) for H2052 and H2452 samples.

Techniques: Sample Prep, Labeling, Isolation, Expressing, Generated

Journal: EBioMedicine

Article Title: A liquid biopsy for detecting circulating mesothelial precursor cells: A new biomarker for diagnosis and prognosis in mesothelioma

doi: 10.1016/j.ebiom.2020.103031

Figure Lengend Snippet: Validation of the microfluidic approach for MPC analysis. (a) Captured efficiency of mice mesothelioma cell line (RN5) and human mesothelioma cell lines (H2052 and H2452) spiked in healthy blood using MesoFind microfluidic chips. (b) Identification of different MPC and leukocytes from blood using fluorescence microscopy after cell entrapment using MesoFind devices. Three differing MPC subtypes (top three) shown here contains different combinations of CD34 and/or CD90 expression and were negative for CD45. Leukocytes shown express CD34- CD90- CD45+ but were generally identified as CD45+ and can have varying expressions of CD34 and CD90 (not shown). All scale bars are 20 um. (c) Schematic of mesothelioma mice model analysis. RN5 mesothelioma cells were injected intraperitoneally (i.p.) and mice were sacrificed between 1 to 6 weeks post-injection. Control mice were injected with saline. The lavage and blood collected from the mice were analyzed using flow cytometry or MesoFind microfluidic analysis followed by fluorescence microscopy for the identification of different MPC populations. Images were created with BioRender. (d) Total MSLN+ CD34+ CD90+ MPC captured from the lavage collected from mice using MesoFind analysis. A general increase in total counts observed from 0 to 6 weeks post-injection. (e) Flow cytometry of MSLN+ CD34+ CD90+ MPC in lavage samples. No significant change was observed after 4-weeks post-injection. No MPC were detected in blood samples using flow cytometric analysis (not shown). (f) Total MSLN+ CD34+ CD90+ MPC captured from the blood of mice using MesoFind analysis. A general increase in total counts observed from 0 to 6 weeks post-injection. (g) Mesothelin expression indices (EI) for MSLN+ CD34+ CD90+ MPC in lavage and blood samples from 0 to 6 weeks post-injection using MesoFind analysis. (h) Flow cytometric gating for MSLN+ cells and CD34+ CD90+ cells in naive mice and (i) 4 weeks RN5 post-injected mice. All analysis was performed in triplicates (n=3). Error bars for Fig. 2 d,f, and g represent standard error of mean. All reported values were compared to values at 0 week (d,e,f) or naïve mice (g) using unpaired t-test, *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Afterwards, cells were then incubated in the dark for 1 hour at room temperature with a cocktail of antibodies containing either 2mg/mL of anti-CD34 AF488 (bs-8996R-A488, Bioss Antibodies, USA), 2mg/mL of anti-CD90 AF555 (bs-0778R-A555, Bioss Antibodies, USA), and 2 mg/mL streptavidin AF647 (405237, Biolegend, USA) for RN5 samples or 1mg/mL of anti-CD34 AF488 (ab195013, Abcam, Canada), 0.5mg/mL of anti-CD90 AF555 (bs-10430R-A555, Bioss Antibodies, USA), and 2 mg/mL streptavidin AF647 (405237, Biolegend, USA) for H2052 and H2452 samples.

Techniques: Fluorescence, Microscopy, Expressing, Injection, Flow Cytometry

Journal: EBioMedicine

Article Title: A liquid biopsy for detecting circulating mesothelial precursor cells: A new biomarker for diagnosis and prognosis in mesothelioma

doi: 10.1016/j.ebiom.2020.103031

Figure Lengend Snippet: Clinical utility of analyzing various MPC subpopulations using MesoFind device. (a) Total blood counts for MSLN+ CD34+ CD90+ MPC from healthy donor (HD, n=10), asbestos-exposed individuals (ASB, n=23), and malignant pleural mesothelioma patients (MPM, n=23) using MesoFind devices. (b) Comparison of mesothelin expression indices between varying subpopulations of MPC (CD90+ CD34-, CD90- CD34+, CD90+ CD34+, CD90+, CD34+) in HD, ASB, and MPM individuals. All MPC were MSLN+ and CD45-. (c-h) MSLN expression indices (EI) for most representative MPC subtypes in each clinical evaluation. Overall, higher MSLN expression indices corresponded to higher severity in clinical tests. EI cut-off values between each group were determined and the clinical sensitivity and specificity were reported (right). (c) EI of MSLN+ CD90- CD34+ CD45- MPC in ASB (n=23) and MPM (n=23). Cut-off EI=500. (d) EI of MSLN+ CD90+ CD34- CD45- MPC in HD (n=10) and ASB (n=23). Cut-off EI=200. (e) EI of MSLN+ CD90+ CD45- MPC were compared between epithelioid (n=16) and biphasic (n=5) cancer subtypes. Cut-off EI = 2650. (f) EI of MSLN+ CD34+ CD45- MPC in patients with low (<6) (n=4) and high (>6) (n=4) SUV scores through PET scans were compared. Cut-off EI=6700. (g) EI of MSLN+ CD90+ CD34- CD45- MPC for patients with unresectable tumors (n=19) and resectable tumors (n=4) were compared. Cut-off EI=200. (h) Presence (n=3) and absence (n=13) of lymph node metastasis was compared through MSLN+ CD34+ CD45- MPC. Cut-off EI=24500. Statistics are performed with unpaired t-tests, *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Afterwards, cells were then incubated in the dark for 1 hour at room temperature with a cocktail of antibodies containing either 2mg/mL of anti-CD34 AF488 (bs-8996R-A488, Bioss Antibodies, USA), 2mg/mL of anti-CD90 AF555 (bs-0778R-A555, Bioss Antibodies, USA), and 2 mg/mL streptavidin AF647 (405237, Biolegend, USA) for RN5 samples or 1mg/mL of anti-CD34 AF488 (ab195013, Abcam, Canada), 0.5mg/mL of anti-CD90 AF555 (bs-10430R-A555, Bioss Antibodies, USA), and 2 mg/mL streptavidin AF647 (405237, Biolegend, USA) for H2052 and H2452 samples.

Techniques: Expressing

Journal: Molecular Medicine Reports

Article Title: Irradiation alters the differentiation potential of bone marrow mesenchymal stem cells

doi: 10.3892/mmr.2015.4539

Figure Lengend Snippet: CFUs of BMSCs following irradiation. Giemsa staining of the BMSCs following irradiation at (A) 0 Gy, (B) 0.5 Gy, (C) 1 Gy, (D) 2 Gy, (E) 5 Gy, (F) 10 Gy. Original magnification, ×20. The graph shows the number of CFUs following irradiation with doses of 0, 0.5, 1, 2, 5 and 10 Gy. Data are expressed as the mean ± standard deviation * P<0.05, compared with the 0 Gy group. BMSC, bone marrow mesenchymal stem cell; CFU, colony-forming unit.

Article Snippet: Rat polyclonal anti-CD34-fluorescein isothiocyanate (1:1,000; cat. no. bs-2038R; FITC; Bioss Biosynthesis Biotechnology Co., Ltd., Beijing, China), rat monoclonal anti-CD29-FITC (1:1,000; cat. no. 555005; BD Biosciences, San Jose, CA, USA), rat polyclonal anti-CD-44-FITC (1:1,000; cat. no. FAB6577G; R&D Systems, Inc., Minneapolis, MN, USA) and rat anti-CD45-FITC (1:500; cat. no. 554877; BD Biosciences) antibodies were added separately, followed by 30 min incubation in the dark at 4°C.

Techniques: Irradiation, Staining, Standard Deviation

Journal: PLoS ONE

Article Title: Harvesting prevascularized smooth muscle cell sheets from common polystyrene culture dishes

doi: 10.1371/journal.pone.0204677

Figure Lengend Snippet: (A) Phase contrast image of bladder SMCs at passage 3. (B) Phase contrast images of EPCs at passages 0 and 3, respectively. (C) Tubular network formation of EPCs on Matrigel. (D) EPCs were identified by ac-LDL endocytosis and UEA-1 binding. Nuclei were stained with DAPI. (E) Immunofluorescence images showing CD34 and VEGFR2 expression in EPCs. Nuclei were stained with DAPI. The scale bars show 100 μm in (A), 200 μm in (B, C), and 50 μm in (D, E).

Article Snippet: In addition, immunofluorescence assay was performed using RBITC conjugated anti-CD34 and FITC conjugated anti-vascular endothelial growth factor receptor 2 (VEGFR2) polyclonal antibodies (Bioss, China).

Techniques: Binding Assay, Staining, Immunofluorescence, Expressing