human vegf concentrations  (R&D Systems)

Journal: International journal of molecular sciences

Article Title: Paracrine and Autocrine Effects of VEGF Are Enhanced in Human eMSC Spheroids.

doi: 10.3390/ijms232214324

Figure Lengend Snippet: Figure 1. VEGF level is upregulated in conditioned medium from 3D spheroids compared with monolayer eMSCs with/without knockdown of VEGF. (a) VEGF concentration in the CM of eMSCs on different passages. (b) Dynamics of accumulation of VEGF protein in the CM of eMSCs on passage 6. (c) Effect of the inhibition (LY) and activation (PS48) of the PI3K/AKT pathway on VEGF secretion. Monolayer and spheroid eMSCs were treated for 72 h with medium containing 1% FBS (Contr) or supplemented with Ly2090314 (LY) or PS48. VEGF quantity was normalized to volume of the cell medium, and the number of cells in the wells is indicated in the axis caption (in (a,b)). Values are expressed as means ± SD (n = 3), * p < 0.05 spheroids vs. monolayer, # p < 0.05 monolayer vs. siVEGF, and & p < 0.05 PS48 vs. Contr and LY. CM: conditioned medium; FBS: fetal bovine serum.

Article Snippet: The protein concentration of VEGF in the CM was measured using the ELISA kit DuoSet Human VEGF (R&D-system, Minneapolis, MN, USA) following the manufacturer’s instructions.

Techniques: Knockdown, Concentration Assay, Inhibition, Activation Assay

Journal: International journal of molecular sciences

Article Title: Paracrine and Autocrine Effects of VEGF Are Enhanced in Human eMSC Spheroids.

doi: 10.3390/ijms232214324

Figure Lengend Snippet: Figure 2. Conditioned medium from 3D spheroids and monolayer eMSCs stimulates proliferation and tube formation of HUVEC. (a) HUVEC growth curves when replacing 50% of the growth medium with CM monolayer, CM spheroids, DMEM/F12, added with 1% FBS (Contr) and added with recombinant VEGF. Values are expressed as means ± SD (n = 3), * p < 0.05 eMSCs-CM vs. Contr. (b) Representative images of HUVEC tube formation on Matrigel taken at 24 h after addition of eMSC-CM. Scale bar 100 µm. (c) Quantification of tube formation using the angiogenesis assay measurement tool of software Fiji. Values are expressed as means ± SD (n = 3), * p < 0.05 CM spheroids vs. CM monolayer. HUVEC: human umbilical vein endothelial cells; CM: conditioned medium; FBS: fetal bovine serum.

Article Snippet: The protein concentration of VEGF in the CM was measured using the ELISA kit DuoSet Human VEGF (R&D-system, Minneapolis, MN, USA) following the manufacturer’s instructions.

Techniques: Recombinant, Angiogenesis Assay, Software

Journal: International journal of molecular sciences

Article Title: Paracrine and Autocrine Effects of VEGF Are Enhanced in Human eMSC Spheroids.

doi: 10.3390/ijms232214324

Figure Lengend Snippet: Figure 5. Exogenous and endogenous VEGF affect the viability and proliferation of eMSCs. (a) Viability of eMSCs after replacing the medium with 1% FBS (Contr) alone and supplemented with VEGF, TGFβ and small molecules (Ly and PS48) for 48 h, % relative to control monolayer eMSCs. (b) Growth curves of eMSCs after dissociation and seeding in the medium with 1% FBS (Contr) alone or supplemented with TGFβ and VEGF respectively. (c) Cell number of eMSCs after treatment with VEGF and TGFβ for 5 days, % vs. control medium alone. Results are means ± SD (n = 3). * p < 0.05; ** p < 0.01. FBS: fetal bovine serum.

Article Snippet: The protein concentration of VEGF in the CM was measured using the ELISA kit DuoSet Human VEGF (R&D-system, Minneapolis, MN, USA) following the manufacturer’s instructions.

Techniques: Control

Journal: International journal of molecular sciences

Article Title: Paracrine and Autocrine Effects of VEGF Are Enhanced in Human eMSC Spheroids.

doi: 10.3390/ijms232214324

Figure Lengend Snippet: Figure 6. Exogenous and endogenous VEGF affects eMSCs migration. (a) Wound healing scratch assay of control monolayer (scrambled) and monolayer VEGF knockdown (siVEGF) eMSCs. The blue lines, generated by the wound healing measurement tool of ImageJ, represent the wound borders. (b) Quantification of scratch closure, migration velocity, µm/h. (c) Representative images of monolayered and dissociated 3D spheroid of eMSCs stained with DAPI, migrated through transwell inserts to VEGF, TGF and control medium alone; (d) Quantification of transwell migration. Scale bar 100 µm. Results are means ± SD (n = 3). * p < 0.05, ** p < 0.01.

Article Snippet: The protein concentration of VEGF in the CM was measured using the ELISA kit DuoSet Human VEGF (R&D-system, Minneapolis, MN, USA) following the manufacturer’s instructions.

Techniques: Migration, Wound Healing Assay, Control, Knockdown, Generated, Staining