ne r d systems quantikine kit catalog mela20  (R&D Systems)

Journal: JCI Insight

Article Title: Thrombomodulin protects against acute vascular and multiorgan injury in sickle cell disease

doi: 10.1172/jci.insight.193884

Figure Lengend Snippet: ( A and B ) TM expression was reduced in the glomerular endothelium relative to CD31 expression ( n = 8; 4 male, 4 female mice per condition) (original magnification, 600×) by the test for linear trend while ( C ) increased concentrations of soluble TM were released in circulation from control to 26 and 48 hours after cell-free hemoglobin exposure ( n = 6; 3 male, 3 female mice per condition; Mann-Whitney U test). ( D ) Circulating concentrations of neutrophil elastase (ELANE) were increased at 26 hours and trended higher at 48 hours after the cell-free hemoglobin challenge ( n = 4; 2 male, 2 female mice per condition; 1-way ANOVA). ( E ) Administration of sivelestat, an ELANE inhibitor, led to retained TM on the vasculature by fluorescent intensity at 48 hours after cell-free hemoglobin challenge (1-way ANOVA) and ( F ) reduced soluble TM released into circulation ( n = 6; 3 male, 3 female mice per condition; Mann-Whitney U test). Mice were challenged with a cell-free hemoglobin dose of 0.24 g/kg, and samples and tissue were harvested 26 and 48 hours later. Scale bars: 50 μm. * P < 0.05, ** P < 0.01.

Article Snippet: Plasma concentrations of hemoglobin (E88-134, Bethyl Laboratories), TM (ab209880, Abcam), neutrophil elastase (MELA20, R&D Systems), activated protein C (ABIN6953414, Antibodies-online Inc.), thrombin-antithrombin complexes (TAT, ab230933, Abcam), VCAM-1 (ab100750, Abcam), vWF (NBP2-68175, Novus Biologicals), VEGF (MMV00, R&D Systems), E-selectin (DY575, R&D Systems), endothelin-1 (DET100, R&D Systems), soluble C5b-9 complex (E-EL-M1129, Elabscience), and cystatin C (EMCST3, Invitrogen) were measured by ELISA.

Techniques: Expressing, Control, MANN-WHITNEY

Journal: JCI Insight

Article Title: Thrombomodulin protects against acute vascular and multiorgan injury in sickle cell disease

doi: 10.1172/jci.insight.193884

Figure Lengend Snippet: ( A and B ) Acute lung injury by histopathology elicited after cell-free hemoglobin challenge was improved with TM rescue at 2 or 24 hours after the challenge ( n = 8, 4 male, 4 female at 2 hours per condition; n = 6, 3 male, 3 female at 24 hours per condition; Mann-Whitney U test) (original magnification, ×400). ( C ) Lung wet-to-dry ratios increased with cell-free hemoglobin challenges and improved with TM rescue ( n = 6, 3 male, 3 female per condition at 2 and 24 hours; Mann-Whitney U test). Biomarkers of ( D ) neutrophil activity (myeloperoxidase [MPO]) and ( E and F ) inflammation (IL-6, TNF-α) increased after cell-free hemoglobin exposure and were improved with TM rescue in lung lysates from the SCD mice ( n = 8, 4 male, 4 female per condition at 2 and 24 hours; 1-way ANOVA). Mice were challenged with cell-free hemoglobin (0.24 g/kg i.v.) and rescued with TM (5 mg/kg s.c. + 1 mg/kg i.v.) under the respective conditions, and samples and tissue were harvested 24 hours after the TM rescue (26 hours for the 2-hour TM rescue and 48 hours for the 24-hour TM rescue after cell-free hemoglobin challenges). Median and IQR values provided in ; * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Plasma concentrations of hemoglobin (E88-134, Bethyl Laboratories), TM (ab209880, Abcam), neutrophil elastase (MELA20, R&D Systems), activated protein C (ABIN6953414, Antibodies-online Inc.), thrombin-antithrombin complexes (TAT, ab230933, Abcam), VCAM-1 (ab100750, Abcam), vWF (NBP2-68175, Novus Biologicals), VEGF (MMV00, R&D Systems), E-selectin (DY575, R&D Systems), endothelin-1 (DET100, R&D Systems), soluble C5b-9 complex (E-EL-M1129, Elabscience), and cystatin C (EMCST3, Invitrogen) were measured by ELISA.

Techniques: Histopathology, MANN-WHITNEY, Activity Assay

Journal: bioRxiv

Article Title: Elastase mediated white matter damage in cerebral small vessel disease: Microglia - neutrophils pas de deux

doi: 10.1101/2024.12.23.630204

Figure Lengend Snippet: (A) ELANE concentration in the mouse corpus callosum at different time points after BCAS surgery evaluated with ELISA. n = 8–9 mice per group. (B) ELANE catalytic activity in mouse corpus callosum homogenate samples at different time points after BCAS surgery measured by spectrophotometry. n = 8–9 mice per group. (C) ELANE expression in microglia was assessed by flow cytometric analysis at 7 days after BCAS or sham surgery. Quantifications of flow cytometry are displayed in the adjacent graph. n = 6–9 mice per group. (D) ELANE expression in the bone marrow, and MBP expression in the corpus callosum determined by western blot at 3 days, 7 days, 14 days, and 28 days after BCAS or sham surgery. β-actin served as an internal loading control. (E) Quantification of the intensity of ELANE expression in the bone marrow performed with ImageJ. n = 3 replicates per group. (F) Plasma ELANE concentration at different time points after BCAS or sham surgery evaluated with ELISA. n = 8–9 mice per group. See also Figure S1.

Article Snippet: ELANE expression in mouse corpus callosum areas and plasma samples was measured using commercial ELISA kits (MELA20, R&D Systems).

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Spectrophotometry, Expressing, Flow Cytometry, Western Blot, Control, Clinical Proteomics

Journal: Blood Advances

Article Title: Deletion of pyruvate dehydrogenase kinases reduces susceptibility to deep vein thrombosis in mice

doi: 10.1182/bloodadvances.2024013199

Figure Lengend Snippet: Combined deletion of PDK2/4 results in reduced neutrophil-mediated platelet aggregation. The extent of (A) cathepsin G and (B) elastase release was measured using enzyme-linked immunosorbent assay in WT or PDK2/4 –/– neutrophils that were stimulated with PMA (50 nM) for 30 minutes. Values are mean ± SEM, n = 5 mice per group. Statistical analysis was performed using 2-way ANOVA followed by Tukey multiple comparisons test. (C) WT platelets were coincubated and stirred with WT or PDK2/4 –/– neutrophils in an aggregometer before the addition of PMA (10 nM). Results are expressed as the percent change in light transmission with respect to the blank (buffer without platelets and neutrophils) set at 100%. Left: the representative aggregation curve is shown. Right: the line graph shows the quantified data for platelet aggregation. Values are mean ± SEM, n = 5 mice per group. Statistical analysis was performed using 2-way ANOVA followed by Šidák multiple comparisons test. Neu, neutrophils; Plt, platelets.

Article Snippet: The elastase enzyme-linked immunosorbent assay kit was from R&D Systems (MELA20).

Techniques: Enzyme-linked Immunosorbent Assay, Transmission Assay