A flow reactor was designed to incorporate the graphite rods. The reactor was prepared using an inexpensive acrylate‐based material that is transparent and allows visual monitoring of the reaction and detection of air bubbles that may form inside the channel. A schematic of the reactor is designed by FreeCAD, parametric 3D modeler (Figure 5 ). The reactor consists of two caps and a cylinder (inner radius: 5 mm) (Figure 5 A, C). The graphite rod (Figure 5 B) had an accessible surface 5.08 cm2 area in the reactor. The caps at both ends of the cylinder position the rods in the middle of the channel, while small grooves in the cap (Figure 5 A) allow the solution to pass through the channel. An Instech P720 peristaltic pump was used to pump the solution through the channel (Figure 5 E). Glucose solutions were purged with oxygen during the operation of the reactor and pumped into the channel at a flow rate of 0.02 ml min−1.
P720 peristaltic pump
Manufactured by Instech
The P720 peristaltic pump is a laboratory equipment designed for controlled fluid transfer. The pump utilizes a peristaltic mechanism to generate consistent flow rates. It is capable of handling a wide range of flow volumes.
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7 protocols using p720 peristaltic pump
1
Graphite-Enabled Flow Reactor for Glucose Electrooxidation
2
Measurement of Myogenic Tone in Isolated Mesenteric Arteries
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Experiments were performed using isolated third- and fourth-order mesenteric arteries using PSS gassed with 21% O2/5% CO2/74% N2 (pH 7.4). Arterial segments 1–2 mm in length were cannulated at each end in a perfusion chamber (Living Systems Instrumentation) continuously perfused with PSS and maintained at 37°C. Intravascular pressure was altered using a Servo pump model PS-200-P (Living systems) and monitored using pressure transducers. Following development of stable myogenic tone, luminal flow was introduced during experiments using a P720 peristaltic pump (Instech). Arterial diameter was measured at 1 Hz using a CCD camera attached to a Nikon TS100-F microscope and the automatic edge-detection function of IonWizard software (Ionoptix). Myogenic tone was calculated as: 100 x (1-Dactive/Dpassive) where Dactive is active arterial diameter and Dpassive is the diameter determined in the presence of Ca2+-free PSS supplemented with 5 mM EGTA.
3
Calcium Imaging of Shear Stress Response
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Cells were loaded with 5 μM Fura2-AM (Thermo Fisher Scientific,
Waltham, MA) at 37 °C for 30 min. Cells were then washed with
Dulbecco’s phosphate-buffered saline and observed under a 40×
objective lens using a Nikon Eclipse Ti-E microscope controlled by
Elements software. Cytosolic calcium was observed by recording Ca2+-bound Fura excitation fluorescence at 340/380 nm and emission
at 510 nm. Baseline Ca2+ was observed for 5 min prior to
data acquisition. Fluid shear stress was then applied to cells utilizing
an Instech P720 peristaltic pump with an inlet and outlet setup. The
fluid was perfused on the glass-bottom plates at a shear stress of
5 dyn/cm2. After each experiment, the maximum calcium signal
was obtained with ATP (10 μM) to confirm cell viability. Conditions
for all experiments were maintained at 37 °C and 5% CO2 in a stage top cage incubator (okoLab, Burlingame, CA). Ca2+ analysis followed a standard calculation as previously described.56 (link)
Waltham, MA) at 37 °C for 30 min. Cells were then washed with
Dulbecco’s phosphate-buffered saline and observed under a 40×
objective lens using a Nikon Eclipse Ti-E microscope controlled by
Elements software. Cytosolic calcium was observed by recording Ca2+-bound Fura excitation fluorescence at 340/380 nm and emission
at 510 nm. Baseline Ca2+ was observed for 5 min prior to
data acquisition. Fluid shear stress was then applied to cells utilizing
an Instech P720 peristaltic pump with an inlet and outlet setup. The
fluid was perfused on the glass-bottom plates at a shear stress of
5 dyn/cm2. After each experiment, the maximum calcium signal
was obtained with ATP (10 μM) to confirm cell viability. Conditions
for all experiments were maintained at 37 °C and 5% CO2 in a stage top cage incubator (okoLab, Burlingame, CA). Ca2+ analysis followed a standard calculation as previously described.56 (link)
4
Calcium Imaging of Shear Stress Response
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Cells were grown on glass-bottom plates. After treatment with the appropriate drug, the cells were incubated with 5 µM Fura-2 AM (TEFLabs, Austin, TX) at 37 °C for 30 minutes. After washing with DPBS, the cells were observed under a 40 × objective lens with a Nikon Eclipse Ti-E microscope. Fura-2 fluorescence images at excitation of 340/380 nm and emission of 510 nm were recorded. After equilibration under the microscope for 20 minutes, baseline calcium was recorded for 2 minutes and experimental data were acquired. Fluid-shear stress was then applied to cells with an Instech P720 peristaltic pump. The perfused fluid was pumped into the cell culture dish and retained at a shear stress of 1 dyne/cm2 (for epithelial cells) or 8 dyne/cm2 (for endothelial cells) with a constant flow rate of about 20 or 160 μl/sec, respectively. At the end of each experiment, the maximum calcium signal was obtained by perfusion of ATP (10 µM) to confirm cell viability. In addition to autofluorescence, both minimum and maximum fluorescence was collected as previously described84 (link). Conditions for all experiments were maintained at 37 °C and 5% CO2 in a stage top cage incubator (okoLab, Burlingame, CA). Calcium analysis was then followed a standard calculation as previously described84 (link).
5
Isolated Mesenteric Artery Pressure-Myogenic Tone
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Experiments were performed using isolated third-order mesenteric arteries using PSS gassed with 21% O2, 5% CO2, and 74% N2 (pH 7.4). Arterial segments 1 to 2 mm in length were cannulated at each end in a perfusion chamber (Living Systems Instrumentation) and continuously perfused with PSS at 37 °C. The arterial diameter was measured at 1 Hz using a CCD camera attached to a Nikon TS100-F microscope and the automatic edge-detection function of IonWizard software (Ionoptix). Intravascular pressure was increased using a Servo pump (model PS-200-P, Living Systems Instrumentation) and measured using pressure transducers. Intraluminal flow was introduced using a P720 peristaltic pump (Instech). The intraluminal flow rate required to apply shear stress was calculated from the internal diameter of individual arteries. Myogenic tone was calculated as 100 × (1 − Dactive/Dpassive), where Dactive is active arterial diameter and Dpassive is the diameter determined in the presence of Ca2+-free PSS supplemented with 5 mM EGTA.
6
Isolated Mesenteric Artery Myogenic Tone
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Experiments were performed using isolated third- and fourth-order mesenteric arteries using PSS gassed with 21% O2/5% CO2/74% N2 (pH 7.4). Arterial segments 1–2 mm in length were cannulated at each end in a perfusion chamber (Living Systems Instrumentation) continuously perfused with PSS and maintained at 37°C. Intravascular pressure was altered using a Servo pump model PS-200-P (Living Systems) and monitored using pressure transducers. Following the development of stable myogenic tone, intraluminal flow was introduced using a P720 peristaltic pump (Instech). The intraluminal flow rate required to apply a specific amount of shear stress to each artery was calculated using internal diameter. Arterial diameter was measured at 1 Hz using a CCD camera attached to a Nikon TS100-F microscope and the automatic edge-detection function of IonWizard software (Ionoptix). Myogenic tone was calculated as: 100×(1−Dactive/Dpassive) where Dactive is active arterial diameter and Dpassive is the diameter determined in the presence of Ca2+-free PSS supplemented with 5 mM EGTA.
7
Mesenteric Artery Myogenic Tone Measurement
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Experiments were performed using isolated third-and fourth-order mesenteric arteries using PSS gassed with 21% O 2 /5% CO 2 /74% N 2 (pH 7.4). Arterial segments 1-2 mm in length were cannulated at each end in a perfusion chamber (Living Systems Instrumentation) continuously perfused with PSS and maintained at 37°C. Intravascular pressure was altered using a Servo pump model PS-200-P (Living systems) and monitored using pressure transducers. Following development of stable myogenic tone, luminal flow was introduced during experiments using a P720 peristaltic pump (Instech). Arterial diameter was measured at 1 Hz using a CCD camera attached to a Nikon TS100-F microscope and the automatic edge-detection function of IonWizard software (Ionoptix). Myogenic tone was calculated as: 100 x (1-D active /D passive ) where D active is active arterial diameter and D passive is the diameter determined in the presence of Ca 2+free PSS supplemented with 5 mM EGTA.
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