24-well cell culture plates (Falcon) were coated with Matrigel (Corning Life Sciences, Corning, NY) and allowed to solidify at 37 °C for 1 hour. Cultured HUVECs were then washed with PBS, trypsinized, centrifuged, and resuspended in a mixture of 50% serum from individual patients with HF, LVAD, or OHT and 50% Endothelial Basal Medium-2 (EBM-2, Lonza) with growth factor additives such that the final concentration of each exogenous growth factor in the serum/EBM-2 mixture was equal to that in EGM-2 (Lonza). This mixture containing 200,000 HUVECs was then gently pipetted into the Matrigel-coated wells and incubated for 18 hours under standard conditions in the presence or absence of an Ang-2 blocking antibody (150 ng/mL, azide-free mouse-anti-human-Ang-2, Adipogen, San Diego, CA), which specifically inhibits binding of Ang-2 to Tie-2 but does not affect binding of Ang-1 to Tie-2. Cultures were then stained with Calcein (Corning) to improve microtube visibility. Microtube formation was assessed by microscopy as described26 (link). Briefly, total tube number in a low power field was quantified (5 fields per well were averaged). All quantifications were performed by technicians who were blinded to patient identity and cohort status.
Calcein
Manufactured by Corning
Calcein is a fluorescent dye used in laboratory applications. It is a chemical compound that emits green fluorescence when exposed to ultraviolet or blue light. Calcein is commonly used for cell viability and cytotoxicity assays, as well as for various imaging and detection techniques.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by Corning (4 mentions)
24 well cell culture plate, by BD (1 mentions)
Ebm 2, by Lonza (1 mentions)
Egm 2, by Lonza (1 mentions)
Evos fl imaging system, by Thermo Fisher Scientific (1 mentions)
Bay11 7082, by Selleck Chemicals (1 mentions)
Growth factor reduced matrigel, by Corning (1 mentions)
6 protocols using calcein
1
HUVEC Tube Formation Assay with Ang-2 Inhibition
2
In vitro Tube Formation Assay
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The in vitro tube formation assay was performed in a 24-well plate using Growth Factor-Reduced Matrigel (Corning, 354234). Briefly, HUVECs were stained with cell-permeable dye, calcein (Corning, 354216), for 30 min and replanted in 24-well plates precoated with 150 μl/well Growth Factor-Reduced Matrigel (Corning, 354234) and in cultured with ML385 (purity: 99.55%, MedChemExpress, HY-100523), TBHQ (purity > 98.0%, MedChem Express, HY-100489), Bay11-7082 (Bay, purity: 99.69%, Selleck, S2913) at 37°C in cell culture incubator. After a 12-h incubation, the formed capillary-like tubes were photographed in randomly chosen fields using a microscope (EVOS FL Imaging System, Thermo Fisher Scientific, Waltham, MA, USA). The tube formation was quantified by manual counting using ImageJ software (National Institutes of Health, Bethesda, Maryland, USA).
3
In vitro Angiogenesis Assay for HUVECs
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The in vitro angiogenic activity of HUVECs was determined by Matrigel tube formation assay. Briefly, after the experimental period described above, HUVECs were stained with cell-permeable dye, calcein (Corning, 354216), for 30 min and replated in 24-well plates precoated with 150 μL/well growth factor–reduced Matrigel (Corning, 354234) and incubated at 37 °C in cell culture incubator. After 12 h of incubation, capillary-like tube formation was observed with a computer-assisted microscope (EVOS, Thermo Fisher Scientific, MA, USA). Tube formation was defined as a tube-like structure exhibiting a length four times its width. The tube length in duplicate wells were counted and averaged using ImageJ software.
4
In Vitro Angiogenic Assay of HUVECs
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The in vitro angiogenic activity of HUVECs was determined by Matrigel tube formation assay. After the experimental period described above, the HUVECs were stained with cell-permeable dye, calcein (Corning, 354216), for 30 min and replated in 24-well plates precoated with 150 μL/well growth factor-reduced Matrigel (Corning, 354234) and incubated at 37℃ in cell culture incubator. After 12 h of incubation, capillary-like tube formation was observed with a computer-assisted microscope (Thermo Fisher Scientific, EVOS). Tube formation was defined as a tube-like structure exhibiting a length four times its width. The tube length in duplicate wells was counted and averaged using ImageJ software.
5
Quantifying In Vitro Angiogenesis in HUVECs
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Matrigel tube formation assays were used to assess the in vitro angiogenic activity of HUVECs. Following the completion of the aforementioned experimental protocol, calcein (Corning, 354216), a cell-permeable dye, was used to stain the HUVECs. After a 30-min incubation, the HUVECs were replated onto Matrigel-precoated 24-well plates (with 150 μL/well of growth factor–reduced Matrigel; Corning, 354234), which were transferred to a 37°C cell culture incubator for 12 h. After incubation, a computer-assisted microscope (EVOS, Thermo Fisher Scientific, MA, United States) was used to assess capillary-like tube formation, as defined by the presence of tube-like structures at least four times as long their widths. The tube lengths in duplicate wells were counted and averaged using ImageJ software.
6
In Vitro Angiogenic Activity Assay
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The in vitro angiogenic activity of HUVECs was determined by Matrigel tube formation assay. After the experimental period described above, the HUVECs were stained with cell-permeable dye, calcein (354216, Corning incorporated, Corning), for 30 min and replated in 24-well plates precoated with 150 μL/well growth factor-reduced Matrigel (354234, Corning incorporated) and incubated at 37°C in cell culture incubator. After 12 h of incubation, capillary-like tube formation was observed with a computer-assisted microscope (EVOS, Thermo Fisher Scientific). Tube formation was defined as a tubelike structure exhibiting a length four times its width. The tube length in duplicate wells was counted and averaged using ImageJ software.
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