Tube formation of HRMECs was tested using a previously described protocol [26] (link). Growth Factor Reduced Matrigel matrix (Corning) (300 μL) was laid on the bottom of a 24-well plate, onto which HRMECs were seeded. Capillary-like structures were observed under a Nikon Eclipse Ti inverted microscope (Nikon) 20 h after cell seeding. At least six different fields were randomly selected and observed in each well. Meshes, branches, and the branching length of the capillary-like structures were analyzed using ImageJ software (version1.49p; NIH, Bethesda, MD, USA).
Growth factor reduced matrigel matrix
Manufactured by Corning
Sourced in United States, Germany
Growth Factor Reduced Matrigel Matrix is a soluble basement membrane extract of proteins derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. It is commonly used as a cell culture substrate to support the growth and differentiation of various cell types.
Automatically generated - may contain errors
Lab products found in correlation
Rat tail collagen type 1, by Corning (2 mentions)
Slides for angiogenesis, by Ibidi (2 mentions)
Cc 3162, by Lonza (2 mentions)
Bz x700 microscope, by Keyence (2 mentions)
Ab134626, by Abcam (2 mentions)
Ad yfp, by Takara Bio (2 mentions)
Human recombinant bfgf, by Thermo Fisher Scientific (2 mentions)
Human recombinant ngf, by Thermo Fisher Scientific (2 mentions)
Fibroblasts medium, by ScienCell (2 mentions)
Epicult b medium, by STEMCELL (2 mentions)
Epicult b medium, by Corning (2 mentions)
Human recombinant mmp3, by Thermo Fisher Scientific (2 mentions)
Mammary epithelial growth medium, by Lonza (2 mentions)
Recombinant human egf, by Thermo Fisher Scientific (2 mentions)
Matrigel basement membrane matrix high concentration, by Corning (2 mentions)
Eclipse ti, by Nikon (1 mentions)
Stereo discovery microscope, by Zeiss (1 mentions)
Thincert, by Greiner (1 mentions)
24 well plate, by Corning (1 mentions)
Ckx41 inverted microscope, by Olympus (1 mentions)
47 protocols using growth factor reduced matrigel matrix
1
Tube Formation Assay for HRMECs
2
Investigating Cell Invasion and Migration Mechanisms
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell invasion assays were performed using transwell migration chambers, as described previously13 (link). Briefly, 4175 and 4T1 cells were pretreated with 1 µM RAGE inhibitors (FPS-ZM1 or TTP488) or 0.1% DMSO for 1 h in culture media. 4175 cells (1.5 × 104 cells) and 4T1 cells (6.5 × 104 cells) were seeded in the upper chamber of 8-μm porous transwell inserts (cat# 662638 ThinCerts; Greiner bio-one) coated with 12.5 μg of Growth Factor Reduced Matrigel matrix (cat# 354230, Corning), in serum-free DMEM or RPMI, and incubated in 24-well plates with 1% FBS as a chemoattractant for 24 h. RAGE inhibitors or DMSO control were added to the appropriate complete media in both the upper and lower chambers. Following incubation for 16 h, cells were fixed with methanol for 10 min and stained with 0.1% crystal violet in dH2O. Non-invaded cells and excess stain were rinsed with water and removed from the inner surface of the insert with a cotton swab. To quantify the invaded cells, the insert membranes were imaged at 2x magnification (SteREO Discovery microscope, Zeiss), and the invaded area % was quantified with ImageJ 1.34n software (National Institutes of Health, USA). To assess cell migration, the same transwell assay method was used without Matrigel coating of the transwell inserts, as we have previously reported42 (link),70 (link).
3
Transwell Invasion Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transwell invasion assays were performed using inserts with 8.0-μm pores (Falcon). 5 × 104 cells were mixed in total 5 mg/ml growth factor reduced Matrigel matrix (Corning) and plated in inserts in a total volume of 30ul, and inserts were placed in 24-well plates (Corning). After incubation for 30 min at 37°C in a 5% CO2 incubator, 200 ul serum-free culture medium was added to the inserts followed by the addition of 500 ul of complete culture media to the bottom wells. After 24 hr, invaded cells that accumulated on the bottom surface of the insert were fixed with 4% paraformaldehyde for 10 min at room temperature, washed with 1× phosphate-buffered saline (PBS) for 10 min. The cells were then fixed with 100% methanol for 10 min at room temperature, washed 3 times with 1× PBS and stained with 0.5% Crystal violet for 30 min. The cells and Matrigel were cleaned out of the inserts using cotton swabs and the membranes were mounted on slides and imaged using an Olympus CKX41 inverted microscope and analyzed using Infinity Analyze 7. The total number of cells/membrane were counted and the experiments were performed each in triplicate.
4
Angiogenic Potential of PBMCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To compare pro-angiogenic properties of PBMCsec, a tube formation assay was performed with HUVECs (passage 4) as described previously.21 (link),25 (link) Cells were isolated as described59 (link) and routinely cultured in endothelial cell growth basal medium-2 (EBM-2; Lonza Group AG, Basel, Switzerland), supplemented with endothelial cell growth medium-2 (EGM-2; BulletKit; Lonza). Prior to the tube formation assay, cells were maintained in EBM-2 containing 2% (vol/vol) heat-inactivated fetal bovine serum (Lonza) overnight and starved in basal EBM-2 for 4 h. Cells were seeded on growth factor-reduced Matrigel Matrix (Corning Life Sciences, Tewksbury, MA, USA) in μ-slides angiogenesis (ibidi, Graefelfing, Germany) at a density of 104 cells/cm2 and stimulated with the supernatant obtained from 4 × 106 PBMCs for 3 h. Micrographs were acquired by an inverted-phase contrast microscope (CKX41; Olympus, Tokyo, Japan) equipped with a 10× objective (CAch N, 10×/0.25 PhP; Olympus) using a SC30 camera (Olympus) and cellSens Entry software (v.1.8; Olympus). Tubule formation was quantified by the Angiogenesis Analyzer plug-in of ImageJ using default settings.60
5
Synthesis and Characterization of Chitosan-Based Biomaterials
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Chitosan [molecular weight (Mw): ~30 × 104 Da, 92% deacetylated] was obtained from Shenzhen Zhongfayuan Biological Technology Co. Ltd. (Shenzhen, China). PSS (Mw: 1.7 × 104 Da) was purchased from Sigma-Aldrich (St. Louis, MO, USA). CCS (Mw: ~1.0 × 105 Da) and HACC (Mw: ~1.0 × 105 Da) were purchased from Yuanye Biotech. Co. Ltd. (Shanghai, China). Growth factor–reduced Matrigel Matrix and rat tail collagen type I were obtained from Corning Inc. (Corning, NY, USA). TRIzol reagent, PrimeScript RT reagent kit, and SYBR Premix Ex Taq were purchased from Takara Biotechnology Co. Ltd. (Dalian, China). Gelatin sponge (GS) was purchased from Xiang’en Medical Technology Development Co. Ltd. (Jiangxi, China). SCS was synthesized as previously described (39 (link)). A FluoroTagTM FITC Conjugation kit (FITC1, Sigma-Aldrich) was used to label SCS with FITC according to the manufacturer’s instructions. All cell culture–related reagents were available from Gibco (Grand Island, NY, USA).
6
Oxidized LDL Induced Endothelial Dysfunction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Growth factor-reduced Matrigel matrix was purchased from Corning (Bedford, MA, USA). FxCycle™ PI/RNase Staining Solution, H2DCFDA, DAF-FM diacetate, and DAPI were bought from Life Technologies Corporation (Eugene, OR, USA). Anhydrous sodium sulfide (Na2S), Pierce BCA protein assay kit, RIPA buffer, protease and phosphatase inhibitor mini tablets, RevertAid RT Reverse Transcription Kit, PowerUp SYBR™ Green Master Mix, TRIzol Reagent and CD68 antibody were procured from Thermo Scientific (Rockford, IL, USA). Na2S solution was prepared in molecular biology grade water. Human oxidized low-density lipoprotein (oxLDL) was acquired from Kalen Biomedical, LLC (Montgomery, MD, USA). Cell proliferation reagent WST-1 was obtained from Roche Diagnostics GmbH (Mannheim, Germany). Phospho-eNOS (Ser1177), phospho-AKT (Ser473), phospho-ERK1/2 (Thr202/Tyr204), total AKT, total ERK1/2, total eNOS, Ki67, and CD45 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). GAPDH antibody was procured from Santa Cruz Biotechnology (Dallas, TX, USA). LYVE-1 antibody was purchased from Abcam (Cambridge, MA, USA). CD31 antibody was obtained from R&D Systems, Inc. (Minneapolis, MN, USA). Ready-to-use intercept blocking buffer to block the nitrocellulose membranes was purchased from Li-Cor Biosciences (Lincoln, NE, USA).
7
In Vitro Vasculogenesis Assessment
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In vitro vasculogenesis or angiogenesis was assessed at passage 4 by plating 1 × 104 cells on 10 µl of growth factor reduced matrigel matrix (Cat. 354,230; Corning) in µ-slides for angiogenesis (IBIDI, Germany). Cells were imaged using phase contrast microscope and 10 × objective after 12 h incubation at 37 °C and 5% CO2. This assay was performed in triplicate and number of tube junctions, number of branches of capillary tube and tube length were analysed using Image J (1.51 J8; National Institute of Health, USA) software with angiogenesis analyser plugin (http://rsb.info.nih.gov/ij/macros/toolsets/Angiogenesis%20Analyzer.txt ) in 6 images per participant obtained from random selected microscopic field.
8
In vitro Angiogenesis Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In vitro vasculogenesis or angiogenesis was assessed by plating 5 × 103 cells on growth factor reduced matrigel matrix (Cat. 354,230; Corning) in µ-slides for angiogenesis (IBIDI, Germany). Cells were imaged using phase contrast microscope after 06 h of incubation at 37 °C and 5% CO2. This assay was performed in triplicate and number of tube junctions, number of branches of capillary tube and tube length were analysed using Image J (version 1.51 J8; National Institute of Health, USA) software with angiogenesis analyser plugin (http://rsb.info.nih.gov/ij/macros/toolsets/Angiogenesis%20Analyzer.txt ).
9
Evaluation of Endothelial Cell Tube Formation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The tube formation ability was determined by performing tube formation assay. Growth Factor Reduced Matrigel matrix (Corning) (300 μL) was put on the bottom of a 24-well plate, and HRMECs (2 × 104 cells/per well) were seeded into wells. After 20 h, capillary-like structures were visualized with a Nikon Eclipse Ti inverted microscope (Nikon). For each well, at least six different fields were randomly chosen for observation. Finally, meshes and branch length of the capillary-like structures were evaluated employing ImageJ software (version1.49p; NIH, Bethesda, MD, United States).
10
Evaluating LNT Effects on Endothelial Tube Formation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tube formation assay was used to evaluate the effect of LNT on endothelial cells as descried previously with minor modifications [23 (link), 24 (link)]. Briefly, human umbilical vein endothelial cells (HUVECs) were cultured in DMEM medium containing 10% FBS. Growth factor reduced matrigel matrix (CORNING) was placed in a refrigerator (4 °C) overnight to thaw the matrigel. Plates (24-well) were coated with 100 μl/well of matrigel matrix and were incubated at 37 °C for 30 min to allow the gel to solidify. HUVECs (30,000 cells/well) were seeded onto the top of the gel and were treated with different concentrations of LNT for 12 h in triplicate at 37 °C with 5% CO2. The process of tube formation was monitored every 3 h and the pictures were taken by using a Box-Type Fluorescence Imaging Device (OLYMPUS). The numbers of the tubes and branches in each well were counted.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!