mouse antibody against flk 1  (Santa Cruz Biotechnology)

Journal: Stem Cell Reports

Article Title: Bone Morphogenetic Protein 9 Regulates Early Lymphatic-Specified Endothelial Cell Expansion during Mouse Embryonic Stem Cell Differentiation

doi: 10.1016/j.stemcr.2018.11.024

Figure Lengend Snippet: BMP9 Increases the Formation of LYVE-1 + Cells during the Co-culture of ESC-Derived Vascular Precursors on Murine Stromal OP9 Cells (A) Flow-cytometry analysis of the formation of LYVE-1 + early lymphatic-specified endothelial cells from co-cultures of FLK-1 + vascular precursors on OP9 stromal cells, in response to variable concentrations of BMPs. Vascular precursors were plated on OP9 stromal cells at day 5, and BMPs were added under serum-free conditions after 24 hr of co-culture. The formation of LYVE-1-positive cells was measured at day 10 (after 4 days of stimulation with BMPs). Data are the mean ± SEM of 4 (BMP2), 8 (BMP10), or 11 (BMP9) different experiments. ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05, significantly different from basal control values using an unpaired Student's t test. (B) Schematic of the differentiation kinetics protocol with the different time points used for pulse labeling with BrdU. (C) Flow-cytometry analysis of the formation of LYVE-1-positive cells in the absence (CTL) or in the presence of 0.3 ng/mL BMP9 added at day 6. The reported data originated from pooled different experiments. They are the mean values ± SEM obtained from 2 (Day6), 5 (Day7 and Day8), and 8 (Day10/11) different sets of values, according to the considered time point. ∗∗∗ p < 0.001, significantly different from its respective control using unpaired Student's t test. (D) Representative flow-cytometry charts of pulse BrdU labeling of the co-cultures after 24 hr (Day 7) and 48 hr (Day 8) without (CTL) or after BMP9 (0.3 ng/mL) stimulation. The upper dot plots illustrate the LYVE-1 cell immunoreactivity (LYVE-1-positive cells are gated in R2), and the lower histogram plots show BrdU immunoreactivity of the R2-gated LYVE-1-positive cell population. The percentage of positive cells is indicated on each plot. The BMP9 fold stimulation of BrdU incorporation in LYVE-1-positive cells is reported in the right histogram chart and presented as the mean ± SD of two independent experiments. See also Figure S2 .

Article Snippet: After staining with a phycoerythrin (PE)-conjugated rat monoclonal antibody against mouse FLK-1 (BD Pharmingen), FLK-1-positive cells were sorted using either a MoFlo FACS cell sorter (DAKO Cytomation, Glostrup, Denmark) or a BD FACS Melody cell sorter (BD Biosciences), and subcultured on an OP9 cell stromal feeder layer as previously described ( ).

Techniques: Co-Culture Assay, Derivative Assay, Flow Cytometry, Labeling, BrdU Incorporation Assay

Journal: Stem Cell Reports

Article Title: Bone Morphogenetic Protein 9 Regulates Early Lymphatic-Specified Endothelial Cell Expansion during Mouse Embryonic Stem Cell Differentiation

doi: 10.1016/j.stemcr.2018.11.024

Figure Lengend Snippet: Low Concentrations of BMP9 Modulate the Calcineurin/NFATc1 Signaling Pathway (A) qRT-PCR analysis of BMP9 effects on the mRNA levels of transcription factors known to be essential for lymphatic differentiation. FLK-1 + vascular precursors at day 5 were plated on CellTracker CMFDA-stained OP9 stromal cells. BMP9 (0.3 ng/mL) was added at day 6. After 24 hr of treatment, the CMFDA-negative cell population was sorted and collected for gene expression analysis. Data are the mean ± SEM of five experiments. (B) NFATc1 protein expression analysis after BMP9 stimulation. FLK-1 + vascular precursors at day 5 were stimulated in suspension in serum-free medium with or without 0.3 ng/mL BMP9 for 20 min. Immunoblotting was performed using an antibody against NFATc1 or actin proteins as a loading control. Results of a representative experiment, out of three, are illustrated. Sizes of the molecular weight markers are indicated on the left. (C) Inhibition of NFATc1 signaling by cyclosporin A prevents the BMP9-induced increase in LYVE-1 + cell formation. LYVE-1-positive cell formation was measured by flow cytometry 4 days after addition of BMP9 in the absence or presence of cyclosporin A. Data are the mean ± SEM of three experiments. ∗ p < 0.05, significantly different from control untreated cells (A) and significantly different from treatment with BMP9 alone (C) using unpaired Student's t test. See also Figure S3 .

Article Snippet: After staining with a phycoerythrin (PE)-conjugated rat monoclonal antibody against mouse FLK-1 (BD Pharmingen), FLK-1-positive cells were sorted using either a MoFlo FACS cell sorter (DAKO Cytomation, Glostrup, Denmark) or a BD FACS Melody cell sorter (BD Biosciences), and subcultured on an OP9 cell stromal feeder layer as previously described ( ).

Techniques: Quantitative RT-PCR, Staining, Expressing, Western Blot, Molecular Weight, Inhibition, Flow Cytometry

Journal: Stem Cell Reports

Article Title: Bone Morphogenetic Protein 9 Regulates Early Lymphatic-Specified Endothelial Cell Expansion during Mouse Embryonic Stem Cell Differentiation

doi: 10.1016/j.stemcr.2018.11.024

Figure Lengend Snippet: Analysis of CD31 + LYVE-1 − Endothelial Cell Formation during the Co-culture of ESC-Derived Vascular Precursors on Murine Stromal OP9 Cells (A) Flow cytometry quantitative measurement of CD31 + LYVE-1 − cell formation after co-culture of FLK-1 + vascular precursors on OP9 stromal cells in response to variable concentrations of BMP9. Data are the mean ± SEM of 4 (BMP2), 8 (BMP10), or 11 (BMP9) different experiments. ∗ p < 0.05, significantly different from basal control values using an unpaired Student's t test. (B) Immunofluorescence staining with anti-CD31 and anti-LYVE-1 antibodies of cell co-cultures at day 10 of differentiation, in the absence (CTL) or presence of 10 ng/mL BMP9. Scale bars, 200 μm.

Article Snippet: After staining with a phycoerythrin (PE)-conjugated rat monoclonal antibody against mouse FLK-1 (BD Pharmingen), FLK-1-positive cells were sorted using either a MoFlo FACS cell sorter (DAKO Cytomation, Glostrup, Denmark) or a BD FACS Melody cell sorter (BD Biosciences), and subcultured on an OP9 cell stromal feeder layer as previously described ( ).

Techniques: Co-Culture Assay, Derivative Assay, Flow Cytometry, Immunofluorescence, Staining

Journal: Stem Cell Reports

Article Title: Bone Morphogenetic Protein 9 Regulates Early Lymphatic-Specified Endothelial Cell Expansion during Mouse Embryonic Stem Cell Differentiation

doi: 10.1016/j.stemcr.2018.11.024

Figure Lengend Snippet: Analysis of Differential BMP Type I Receptor Involvement in the BMP9 Low-Dose and High-Dose Effects by RNA-Silencing Experiments (A) Effect of Alk1 and Alk2 siRNA on LYVE-1-positive cell formation induced by 0.3 ng/mL BMP9 in FLK-1 + vascular precursor co-cultures on mouse OP9 cells. Alk knockdowns were performed by siRNA transfection of vascular precursors by either electroporation or by using Lipofectamine RNAiMAX, 24 hr before BMP9 stimulation. Data are the mean ± SEM of three (si Alk2 ) or four (si Scr , si Alk1 ) different experiments. (B) High-dose BMP9-induced LYVE-1 − endothelial cell formation is inhibited after prior Alk2 knockdown in OP9 cells. OP9 cells were first transfected by Lipofectamine RNAiMAX with the different siRNAs, 24 hr before FLK-1 + vascular precursor plating in co-culture on OP9 cells and 48 hr before stimulation with 10 ng/mL BMP9. LYVE-1 − CD31 + cell formation is illustrated. Data are the mean ± SEM of three experiments. (C and D) Alk2 knockdown in OP9 cells prevents SMAD1/5 phosphorylation and VEGF-A secretion induced by 10 ng/mL BMP9. OP9 cells were transfected with the different siRNAs by Lipofectamine RNAiMAX, 48 hr before stimulation with 10 ng/mL BMP9. For SMAD1/5 phosphorylation studies, serum-starved subconfluent OP9 cells were stimulated for 1 hr in the absence (CTL) or presence of BMP9, before immunoblotting with anti P-SMAD1/5 and actin antibodies (C). VEGF-A levels were measured after 48 hr of treatment with or without 10 ng/mL BMP9, by ELISA (D). Data are mean ± SEM of three experiments. ∗ p < 0.05, ∗∗∗ p < 0.001, significantly different from BMP9 stimulation obtained after transfection with siScramble (si Scr ), using unpaired Student's t test. See also Figure S4 .

Article Snippet: After staining with a phycoerythrin (PE)-conjugated rat monoclonal antibody against mouse FLK-1 (BD Pharmingen), FLK-1-positive cells were sorted using either a MoFlo FACS cell sorter (DAKO Cytomation, Glostrup, Denmark) or a BD FACS Melody cell sorter (BD Biosciences), and subcultured on an OP9 cell stromal feeder layer as previously described ( ).

Techniques: Transfection, Electroporation, Co-Culture Assay, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Stem Cell Reports

Article Title: Bone Morphogenetic Protein 9 Regulates Early Lymphatic-Specified Endothelial Cell Expansion during Mouse Embryonic Stem Cell Differentiation

doi: 10.1016/j.stemcr.2018.11.024

Figure Lengend Snippet: Hypothetical Model for BMP9 Regulation of ESC-Derived Vascular Precursor Differentiation in Co-culture with Murine OP9 Stromal Cells Low BMP9 concentrations induce the formation of LYVE-1 + early lymphatic-specified endothelial cells through ALK1 activation. BMP9 was observed to directly activate FLK-1 + vascular precursors and earlier stages of vascular committed cells through mobilization of the NFATc1 signaling pathway. Moreover, an increase in the proliferation of the LYVE-1-positive cell population was found to largely contribute to its expansion. At higher doses, BMP9 could, in addition to ALK1, also activate ALK2 present on OP9 stromal cells. This leads to a significant rise in the secretion of VEGF-A by OP9 cells, with as a resultant effect an increase in LYVE-1 − endothelial cell population at the expense of the LYVE-1 + early lymphatic-specified endothelial cell formation.

Article Snippet: After staining with a phycoerythrin (PE)-conjugated rat monoclonal antibody against mouse FLK-1 (BD Pharmingen), FLK-1-positive cells were sorted using either a MoFlo FACS cell sorter (DAKO Cytomation, Glostrup, Denmark) or a BD FACS Melody cell sorter (BD Biosciences), and subcultured on an OP9 cell stromal feeder layer as previously described ( ).

Techniques: Derivative Assay, Co-Culture Assay, Activation Assay

Journal: Experimental and Therapeutic Medicine

Article Title: Whole bone marrow cell culture: A convenient protocol for the in vitro expansion of endothelial progenitor cells

doi: 10.3892/etm.2014.1827

Figure Lengend Snippet: Whole bone marrow cell (WBMC) culture for endothelial progenitor cells (EPCs) and their identification in vitro . (A) Following two days of culture, a small ‘blood island’ surrounded by spindle-like cells was observed. (B) Following seven days of culture, cells exhibited a spindle-like appearance. The majority of the cells were immunopositive for (C) 1,10-dioctadecyl-3,3,30,30-tetramethylindocarbocyanine perchlorate-labeled acetylated low-density lipoprotein (DiI-acLDL) and (D) FITC-conjugated Flk-1. (E) Cells positive for FITC-lectin and DiI-acLDL were recognized as EPCs undergoing differentiation. (F) Following cell culture for ~2 weeks, endothelial colony-forming cells, which originated from the EPCs, appeared and exhibited a typical cobblestone morphology. Cells expressed CD34 and Flk-1 in the (G) WBMC and (H) bone marrow mononuclear cell culture systems. FITC, fluorescein isothiocyanate; Flk-1, fetal liver kinase 1; CD, cluster of differentiation. WBMC culture for EPCs and their identification in vitro . Comparison between WMBC-EPCs and BMMC-EPCs with regard to the expression levels of (I) double fluorescence positive cells for DiI-acLDL and FITC-Flk-1, (J) CD34, (K) Flk-1 and (L) double fluorescence positive cells for CD34 and Flk-1, and (M) the time that the first endothelial colony-forming cells appeared. Data (n=6) are presented as the mean ± standard deviation. * P>0.05. WBMC, whole bone marrow cell; BBMC, bone marrow mononuclear cell; EPCs, endothelial progenitor cells; DiI-acLDL, 1,10-dioctadecyl-3,3,30,30-tetramethylindocarbocyanine perchlorate-labeled acetylated low-density lipoprotein; FITC, fluorescein isothiocyanate; Flk-1, fetal liver kinase 1; CD, cluster of differentiation.

Article Snippet: The cells were incubated for 20 min in the dark at 4°C with 2 μl FITC-conjugated rat monoclonal antibody against mouse Flk-1 (BD Biosciences) and 5 μl phycoerythrin (PE)-conjugated rat monoclonal antibody against mouse CD34 (BD Biosciences).

Techniques: In Vitro, Labeling, Cell Culture, Expressing, Fluorescence, Standard Deviation

Journal: Current Pharmaceutical Design

Article Title: New Pharmaceutical Treatment of Gastric MALT Lymphoma: Anti-angiogenesis Treatment using VEGF Receptor Antibodies and Celecoxib

doi: 10.2174/13816128113199990420

Figure Lengend Snippet: VEGF-C and Flt-4 Immunoreactivity in the H. heilmannii -treated mouse fundic mucosa. a , b : VEGF-C immunoreactivity is recognized in the marginal area of the lymphoma. ( a : x200, b : x400) c , d : Flt-1 immunoreactivity is found mostly in the marginal area of the lymphoma. ( c : x200, d : x 1000)

Article Snippet: Rabbit polyclonal antibodies against Flt-1, Flk-1and Flt-4 (50 mg/mouse) (R&D Systems, MN, USA) were intraperitoneally administered.

Techniques:

Journal: Current Pharmaceutical Design

Article Title: New Pharmaceutical Treatment of Gastric MALT Lymphoma: Anti-angiogenesis Treatment using VEGF Receptor Antibodies and Celecoxib

doi: 10.2174/13816128113199990420

Figure Lengend Snippet: Statistical analysis of the effect of Flt-4 on MALT lymphoma. a : Time course of the change of surface area occupied by MALT lymphoma through stereomicroscopic observation. Mixed administration of Flt-4 and Flk-1 significantly decreased the occupied area in four weeks. A P value less than 0.05 denoted the presence of a statistically significant difference. b : Time course of the size of MALT lymphoma by light microscopic observation. The tumor showed a tendency to shrink, especially following the mixed administration of Flt-4 and Flk-1. c : Time course of the caspase-3-immunoreactive cells within the tumor by confocal laser microscopy. The number of caspase-3-immunoreactive cells in MALT lymphoma was greatest one week after in all Flt-1 administered group.

Article Snippet: Rabbit polyclonal antibodies against Flt-1, Flk-1and Flt-4 (50 mg/mouse) (R&D Systems, MN, USA) were intraperitoneally administered.

Techniques: Microscopy

Journal: Current Pharmaceutical Design

Article Title: New Pharmaceutical Treatment of Gastric MALT Lymphoma: Anti-angiogenesis Treatment using VEGF Receptor Antibodies and Celecoxib

doi: 10.2174/13816128113199990420

Figure Lengend Snippet: Caspase-3 and prox-1 immunoreactivities in H. heilmannii -infected and Flt-4- and Flk-1-administered mice. a : One week after the administration, many cells within the MALT lymphoma showed positive immunoreactivity against caspase-3.(x200) b : Three weeks after the administration, the lymphoma became very small and few caspase-3-immunoreactive cells were observed in the lymphoma. (x400) c - e : In double staining with caspase-3 and prox-1, some of the prox-1-positive cells were found to be caspase-3 immunoreactive (arrow). c : Prox-1 immunoreactivity, d: caspase-3 immunoreactivity, e: merged view of c and d. (x1000)

Article Snippet: Rabbit polyclonal antibodies against Flt-1, Flk-1and Flt-4 (50 mg/mouse) (R&D Systems, MN, USA) were intraperitoneally administered.

Techniques: Infection, Double Staining