antibodies for cd133  (Bioss)

Journal: Scientific Reports

Article Title: Clonally selected primitive endothelial cells promote occlusive pulmonary arteriopathy and severe pulmonary hypertension in rats exposed to chronic hypoxia

doi: 10.1038/s41598-020-58083-7

Figure Lengend Snippet: Isolation and characterization of EC clones from CD117 + ECs. ( A ) Isolation and clonal enrichment flow diagram for EC clones from the lungs of EGFP + rats. ( B ) Representative differential interference contrast (DIC) image shows the typical endothelial cobblestone morphology of EC clones. ( C ) Representative DIC image of 2D matrigel assay. ( D ) Representative optical section (confocal microscopy) demonstrate binding of Griffonia simplicifolia lectin (red pseudo colour) in EC clones, indicating a microvascular phenotype (scale bar 25 μm). ( E ) 24 h 3D tube formation assay in fibrin shows formation of tube networks by EC clones (scale bar 200 μm). Cells were visualized by phalloidin staining of actin filaments (red pseudo colour). ( F ) confocal imaging of Matrigel plug at day 14 with EC clones stained for GFP (green pseudo colour) demonstrating GFP + blood vessels. The red autofluorescence demonstrates blood and indicates active perfusion of the GFP + vascular structures after 14 days. Scale bar: 25 μm. Counterstaining in ( D – F ) with DAPI. ( G , H ) Representative images demonstrate that EC clones form free-floating spheroids in low adhesion cell culture wells. When overlaid with a 50Vol%/50Vol% EGM2/Matrigel mixture, EC clone spheroids underwent angiogenic sprouting. The image ( G ) shows green fluorescence channel of EC clone spheroids (green pseudo colour), whereas image ( H ) shows DIC brightfield image of an EC clone spheroid after 7 days of sprouting. Scale bars: 50 μm. ( I ) Representative flow cytometry analysis of EC clones shows expression of endothelial markers (vWF, CD144, VEGFR2, CD105, CD34) and CD117, but not of typical hematopoietic and myeloid markers (CD45, CD11b/c and CD133). Note that vWF is an intracellular marker and required permeabilization of the cells during the staining process. ( J ) The fraction of clonally expandable wells increases from clonal generation 1 to 3. n = 3 (1st) and 6 (2nd, 3rd, 4th). ( K ) 4th generation EC clones proliferate faster than non-expanded CD117 + ECs (n = 3–4). * P < 0.05, ** P < 0.01.

Article Snippet: EC clones and CD117 − control ECs were stained with fluorescent label-conjugated antibodies against CD144 (bs-0878R, Bioss, Woburn, MA), VEGFR2 (bs-05665R, Bioss), CD105 (bs-4909M, Bioss), CD34 (sc-7324, Santa Cruz Biotechnology, Santa Cruz, CA), CD117 (bs-0672R, Bioss), CD45 (202212, Biolegend, San Diego, CA), CD11b/c (554862, BD, Franklin Lakes, NJ) and CD133 (bs-0209R, Bioss) according to previously published standard protocols .

Techniques: Isolation, Clone Assay, Matrigel Assay, Confocal Microscopy, Binding Assay, Tube Formation Assay, Staining, Imaging, Cell Culture, Fluorescence, Flow Cytometry, Expressing, Marker

Journal: PLoS ONE

Article Title: Endothelial cells are a source of Nestin expression in Pulmonary Arterial Hypertension

doi: 10.1371/journal.pone.0213890

Figure Lengend Snippet: ( A ) Representative flow cytometry of rat lung ECs for CD144 (Vascular Endothelial-cadherin) and VEGFR2. Rat lung ECs were negative for myeloid/hematopoietic markers CD133 and CD11b/c. The specific antibody staining is red, and the corresponding isotype is grey. ( B ) Rat lung ECs bind Griffonia simplicifolia lectin (G.s.), indicating microvascular ECs. ( C ) Rat lung ECs grown on chamber slides express Nestin. Note the perinuclear localization and the filaments extending throughout the cytoplasm. Control means omission of primary antibody. (B-C): Scale bars: 50 μm. ( D ) Representative Western blot showing Nestin expression in HLMVECs (β-actin as loading control). ( E ) Representative Western blots showing PCNA expression in HLMVECs (α-tubulin as loading control). Experiments 1–3 indicate unstimulated cells grown in separate dishes in EGM-2MV for Western blot analysis.

Article Snippet: Expression of myeloid/hematopoietic markers CD133 (bs-0209R, Bioss) and CD11b/c (554862, BD Biosciences) was excluded by flow cytometry.

Techniques: Flow Cytometry, Staining, Western Blot, Expressing