mouse vegf duoset elisa  (Bio-Techne corporation)

Journal: bioRxiv

Article Title: Signaling events at TMEM doorways provide potential targets for inhibiting breast cancer dissemination

doi: 10.1101/2024.01.08.574676

Figure Lengend Snippet: A) VEGF ELISA of conditioned media obtained from BAC1.2F5 macrophages (Mϕ) with CSF-1 (3000units/mL) for the times indicated. VEGF is indicated as absolute amount (pg/ml) of secreted protein. n=3 individual experiments done in duplicate, *p<0.05, by two-way ANOVA. B) CSF-1 ELISA of conditioned media obtained from MDA-MB 231 tumor cells (TC). TCs were cultured for 24 hours in serum-free media and amount of CSF-1 secreted by the TCs measured in the tumor cell conditioned media by ELISA. Control is media not exposed to tumor cells but treated the same way as the cells. CSF1 secreted is denoted in absolute amount (pg/mL) of secreted protein. n=3 individual experiments done in duplicate. *p<0.05 ****p<0.0001 by two-way ANOVA. C) Immunofluorescence staining and quantification of PyMT tumor sections stained for CSF-1R (green), vasculature (CD31, magenta), TMEM doorway macrophages (CD206, red) and DAPI (blue). The cells composing a TMEM doorway, identified as described in , are indicated by the points of the yellow triangle being the relative positions of the TMEM doorway endothelial cell (TEC, white circle), TMEM doorway macrophage (TM, blue circle) and TMEM doorway tumor cell (TTC, pink circle) indicated with white arrows. Scale bar 50 μm. D) Quantification of the percent of CD68+ TMEM doorway macrophages and CD206+ TMEM doorway macrophages which stain positively for CSF-1R ( n = 6).

Article Snippet: ELISA was performed as per the manufacturer’s recommendation using the mouse VEGF DuoSet ELISA kit from R&D Systems.

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Control, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Signaling events at TMEM doorways provide potential targets for inhibiting breast cancer dissemination

doi: 10.1101/2024.01.08.574676

Figure Lengend Snippet: A) VEGF ELISA of conditioned media obtained from macrophages (BAC1.2F5) co-cultured with MDA-MB-231 tumor cells treated with control antibody (ctrl Ab), CSF-1R blocking Ab or CSF-1R inhibitor (GW2580), denoted in absolute amount (pg/mL) of secreted protein. n=3 individual experiments done in duplicate, *p<0.05, ***p<0.001, ****p<0.0001 analyzed by two-way ANOVA. B) VEGF ELISA of conditioned media obtained from macrophages (BAC1.2F5) cultured in conditioned media from MDA-MB-231 tumor cells and treated with control Ab, CSF-1R blocking Ab (CSF-1R Ab) or CSF-1R inhibitor (GW2580), denoted in absolute amount (pg/mL) of secreted protein. n=3 individual experiments done in duplicate, *p<0.05, **p<0.01, ****p<0.0001 analyzed by two-way ANOVA. C) VEGF ELISA of conditioned media obtained from bone marrow macrophage (BMMs) either expressing or lacking CSF-1R co-cultured with MDA-MB-231 tumor cells for the times indicated. VEGF levels are denoted in absolute amount (pg/mL) of secreted protein. n=3 individual experiments done in duplicate, ****p<0.0001 analyzed by two-way ANOVA. D) VEGF ELISA of conditioned media obtained from BMMs either expressing or lacking CSF-1R cultured in conditioned media from MDA-MB-231 tumor cells for the times indicated. VEFG levels are denoted in absolute amount (pg/mL) of secreted protein. n=3 individual experiments done in duplicate, ****p<0.0001 analyzed by two-way ANOVA. E) Immunofluorescence staining of VEGF in macrophages (labelled with CellTracker ™ Green) cultured with or without tumor cells (labelled with CellTracker ™ Red) and treated with either ctrl Ab or CSF-1R blocking Ab. Scale bar 5 μm. F) Quantitation of the VEGF fluorescence intensity in macrophages in E. The amount of VEGF in the macrophage was quantified using ImageJ. n=3. *p<0.05, **p<0.01, ****p<0.0001 analyzed by two-way ANOVA. G, H) Immuno-staining and quantification of VEGF intensity around TMEM doorways, obtained from PyMT mice treated with control antibody (ctrl Ab) or CSF-1R blocking Ab. Sequential tumor sections derived from PyMT tumors were stained by immunofluorescence (VEGF, Iba-1) and IHC (TMEM doorways-Mena, Iba-1, endomucin). TMEM doorways were identified as described in . The circle in the IHC (black) and IF (white) panels show the same TMEM doorways obtained from the alignment of serial sections, and the three cells making up the TMEM doorway are pointed out with the yellow triangle in each panel. TMEM doorways are outlined in the circle with the endothelial cell (TE, blue stain, white circle), macrophage (TM, brown stain, teal circle) and TMEM doorway tumor cell (TTC, pink stain, pink circle) indicated with arrows. Next, the two sequential sections were aligned and TMEM doorway was identified in the IF-stained section. The immunofluorescence intensity of VEGF-A expression (green stain) in the identified TMEM doorway ROIs was measured and plotted here. N=analysis of images obtained from tissue sections from individual 11 mice, Scale bar 20 μm, not significant, analyzed by t-test.

Article Snippet: ELISA was performed as per the manufacturer’s recommendation using the mouse VEGF DuoSet ELISA kit from R&D Systems.

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Control, Blocking Assay, Expressing, Immunofluorescence, Staining, Quantitation Assay, Fluorescence, Immunostaining, Derivative Assay

Journal: bioRxiv

Article Title: Signaling events at TMEM doorways provide potential targets for inhibiting breast cancer dissemination

doi: 10.1101/2024.01.08.574676

Figure Lengend Snippet: A) In the primary tumor, macrophages and tumor cells exhibit a CSF1-EGF paracrine interaction between tumor cells and macrophages which facilitates tumor cell migration toward blood vessels in breast cancer. Tumor cells secrete CSF-1 which attracts CSF-R1 expressing macrophages, which in turn secrete EGF, increasing the migration of EGFR-expressing tumor cells. The tumor cells within the loop encounter a HGF signal found in a gradient in the tumor microenvironment with the highest concentration near blood vessels. The tumor cells will migrate toward the blood vessels along the HGF gradient, adding directionality to the migration of the tumor cell-macrophage paracrine loop engaged partners. B) In an inactive TMEM doorway (box), with the absence of any CSF1 secreted by the TMEM doorway tumor cell (TTC), the VEGF-A remains within the TMEM doorway macrophage (TM). The blood vessels, including the TMEM doorway endothelial cell (TEC) within the tumor remain sealed and do not allow tumor cells to intravasate through TMEM doorways. C) In contrast, in an active TMEM doorway, CSF-1 is secreted by the TMEM doorway tumor cell and binds to macrophage CSF-1R, stimulating the TMEM doorway macrophage to secrete its VEGF. VEGF causes vascular opening (TAVO event) and allows tumor cells to intravasate through the TMEM doorway, creating CTCs, and dextran or blood to leak out of the vessel. D) These circulating tumor cells (CTCs) travel through the vasculature to secondary sites where TMEM doorways are also found in metastatic foci in lymph nodes and lungs. Thin membranous connections between macrophages and tumor cells stretch from the macrophage on extravascular side of the blood vessel through the endothelial junctions and interact with CTCs which could facilitate CTC extravasation at secondary sites. TMEM doorways in metastases could also be the sites were tumor cells re-intravasate and then seed tertiary metastases.

Article Snippet: ELISA was performed as per the manufacturer’s recommendation using the mouse VEGF DuoSet ELISA kit from R&D Systems.

Techniques: Migration, Expressing, Concentration Assay

Journal: Journal of functional biomaterials

Article Title: Microporous Hydroxyapatite-Based Ceramics Alter the Physiology of Endothelial Cells through Physical and Chemical Cues.

doi: 10.3390/jfb14090460

Figure Lengend Snippet: Figure 3. Expression of angiogenesis-related markers. (A) Dosage of VEGF-A secreted by C166 cells cultured at the surface of HA-based ceramics or plastic culture wells evaluated by ELISA. n = 3 independent experiments. Statistical analysis: two-way ANOVA followed by Tukey’s post hoc test. *: p ≤0.05; **: p ≤0.005. (B) Evaluation of expression of MMP-9 in C166 cells cultured at the surface of HA-based ceramic pellets by western-blot. Upper panel: representative MMP-9-related and actin-related signals. Lower panel: quantification by densitometry of pro- and active MMP-9 signals normalized on the signal detected for actin as housekeeping protein. N = 3 independent experiments. Statistical analysis: two-way ANOVA followed by Tukey’s post hoc test. No significant differences were detected between any of the conditions (p > 0.05).

Article Snippet: ELISA was performed on the culture supernatants according to the manufacturer’s instructions (DuoSet ELISA Development System, Mouse VEGF, R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot