primary mouse antibody against e selectin p selectin  (R&D Systems)

Journal: medRxiv

Article Title: Endothelial cell-activating antibodies in COVID-19

doi: 10.1101/2021.01.18.21250041

Figure Lengend Snippet: A , Schematic workflow for in-cell ELISA. HUVEC were cultured for 6 hours with serum from either healthy controls (collected pre-pandemic) (n=38) or patients hospitalized with COVID-19 (n=118). Cells were then fixed and surface expression of E-selectin ( B ), VCAM-1 ( C ), or ICAM-1 ( D ) was quantified. Median values are indicated by horizontal lines. Groups were analyzed by Mann-Whitney test; ****p<0.0001. E , Beyond the 118 COVID-19 patients tested in panel D, surface expression of ICAM-1 was tested in the context of an additional 126 unique patient samples. Patients requiring mechanical ventilation (n=101) are compared to hospitalized patients who were not mechanically ventilated (n=143); p<0.01 by Mann Whitney test. F , Serum from healthy controls (n=38) and COVID-19 patients (n=102) were assessed for soluble E-selectin. COVID-19 samples were compared to controls by Mann-Whitney test; ****p<0.0001. G , Soluble E-selectin was compared to HUVEC E-selectin expression as presented in panel B. Correlation was determined by Spearman’s method. H , HUVEC were cultured for 6 hours with plasma from healthy controls (n=36), intensive care unit patients with non-COVID sepsis (n=100), or patients hospitalized with COVID-19 (n=72). Cells were then fixed and surface expression of ICAM-1 was quantified. Groups were analyzed by one-way ANOVA with correction for multiple comparisons by Holm-Sidak’s method; *p<0.05 and ***p<0.001.

Article Snippet: After washing with PBS, cells were incubated with 5 µg/ml primary mouse anti-human antibodies against E-selectin (catalog BBA26, R&D), VCAM-1 (catalog BBA5, R&D), or ICAM-1 (ab2213, Abcam) at 4°C overnight.

Techniques: In-Cell ELISA, Cell Culture, Expressing, MANN-WHITNEY

Journal: medRxiv

Article Title: Endothelial cell-activating antibodies in COVID-19

doi: 10.1101/2021.01.18.21250041

Figure Lengend Snippet: HUVEC were cultured for 6 hours with serum from either healthy controls (collected pre-pandemic) (n=38) or patients hospitalized with COVID-19 (n=118). Cells were then fixed and surface expression of E-selectin ( A ), VCAM-1 ( B ), or ICAM-1 ( C ) was quantified. Median values are indicated by horizontal lines. Data were presented as raw absorbance (optical density 650 nm). Groups were analyzed by Mann-Whitney test; ****p<0.0001.

Article Snippet: After washing with PBS, cells were incubated with 5 µg/ml primary mouse anti-human antibodies against E-selectin (catalog BBA26, R&D), VCAM-1 (catalog BBA5, R&D), or ICAM-1 (ab2213, Abcam) at 4°C overnight.

Techniques: Cell Culture, Expressing, MANN-WHITNEY

Journal: medRxiv

Article Title: Endothelial cell-activating antibodies in COVID-19

doi: 10.1101/2021.01.18.21250041

Figure Lengend Snippet: Soluble E-selectin in COVID-19 serum was compared to laboratory and clinical data when available on the same day as the serum collection. Spearman’s correlations are presented for C-reactive protein (n=83) ( A ), D-dimer (n=72) ( B ), calprotectin (n=102) ( C ), oxygenation efficiency (n=99) (pulse oximetry/fraction of inspired oxygen, D ).

Article Snippet: After washing with PBS, cells were incubated with 5 µg/ml primary mouse anti-human antibodies against E-selectin (catalog BBA26, R&D), VCAM-1 (catalog BBA5, R&D), or ICAM-1 (ab2213, Abcam) at 4°C overnight.

Techniques:

Journal: medRxiv

Article Title: Endothelial cell-activating antibodies in COVID-19

doi: 10.1101/2021.01.18.21250041

Figure Lengend Snippet: A , Serum was pooled from 3 patients with positive aCL IgG or 5 patients with positive aPS/PT IgG. HUVEC monolayers were then treated with 2.5% COVID or control serum for 6 hours. Calcein-AM-labeled neutrophils were then added as described in Methods. Scale bar=200 microns. Mean ± standard deviation is presented for n=3 independent experiments; *p<0.05 by one-way ANOVA corrected by Dunnett’s test. B-D , IgG was depleted from each of the aforementioned pools. Activation of HUVEC was determined after culture for 6 hours as defined by surface expression of E-selectin ( B ), VCAM-1 ( C ), or ICAM-1 ( D ). The experiment was repeated on 3 different days, and bars represent mean and standard deviation. Groups were compared by 2-sided paired t-test; *p<0.05 and **p<0.01. E , IgG was purified from the pooled samples referenced in A-C, and then supplemented (100 μg/ml) into control serum that had been depleted of IgG. Activation of HUVEC was determined after culture for 6 hours as defined by surface expression of ICAM-1. Groups were compared by one-way ANOVA with correction for multiple comparisons by Tukey’s test; **p<0.01.

Article Snippet: After washing with PBS, cells were incubated with 5 µg/ml primary mouse anti-human antibodies against E-selectin (catalog BBA26, R&D), VCAM-1 (catalog BBA5, R&D), or ICAM-1 (ab2213, Abcam) at 4°C overnight.

Techniques: Labeling, Standard Deviation, Activation Assay, Expressing, Purification

Journal: International Journal of Environmental Research and Public Health

Article Title: Cytokines and Adhesion Molecules Expression in the Brain in Human Cerebral Malaria

doi:

Figure Lengend Snippet: (original magnification, X400) : Immunohistology for ICAM-1, VCAM-1 & E-selectin. (A–B) Sections of non-malaria cases: (A) Staining for ICAM-1: positive in < 25% of brain microvessel (average score +/−). (B) Staining for VCAM-1: positive in < 25% of brain microvessels (average score +/−). (C) Section of typhoid perforation and septicaemia case: Staining for ICAM-1: positive in 25 to 50% of microvessels (average score +). (D–F) Sections of malaria cases: (D) Staining for ICAM-1: positive in > 75% of microvessels (average score +++). (E) Staining for ICAM-1: positive in > 75% of microvessels (average score +++). Note the more extreme heterogeneity between sequestration and receptor expression in the SMA case in 1E than the CM case in 1D. (F) Staining for ICAM-1: Positive in > 75% of microvessels (average score +++). Note the more intense staining of the CM cerebellar section in 1F compared to the CM cerebral section in 1D.

Article Snippet: Primary antibodies against ICAM-1 (mouse anti-human, monoclonal, Immunotec, UK; dilution 1 : 500), VCAM-1 (mouse anti-human, monoclonal, DAKO, UK; dilution 1 : 200), E-Selectin (mouse anti-human, monoclonal, DAKO, UK; dilution 1 : 200), TNF-α (mouse anti-human, monoclonal, Serotec, UK; dilution 1 : 250), IL-1β (mouse anti-human, monoclonal, Serotec, UK; dilution 1 : 250) and TGF-β (mouse anti-human, monoclonal, Serotec, UK; dilution 1 : 250) were applied for 60 minutes at room temperature.

Techniques: Staining, Expressing

Journal: International Journal of Environmental Research and Public Health

Article Title: Cytokines and Adhesion Molecules Expression in the Brain in Human Cerebral Malaria

doi:

Figure Lengend Snippet: Quantitation of Cerebral Vessels and Co-localization of Sequestration with Expression of Receptors in the 15 Malaria Cases.

Article Snippet: Primary antibodies against ICAM-1 (mouse anti-human, monoclonal, Immunotec, UK; dilution 1 : 500), VCAM-1 (mouse anti-human, monoclonal, DAKO, UK; dilution 1 : 200), E-Selectin (mouse anti-human, monoclonal, DAKO, UK; dilution 1 : 200), TNF-α (mouse anti-human, monoclonal, Serotec, UK; dilution 1 : 250), IL-1β (mouse anti-human, monoclonal, Serotec, UK; dilution 1 : 250) and TGF-β (mouse anti-human, monoclonal, Serotec, UK; dilution 1 : 250) were applied for 60 minutes at room temperature.

Techniques: Quantitation Assay, Expressing