A high-capacity, automated perifusion system was used to dynamically measure hormone secretion from pancreatic islets (Biorep Perifusion V2.0.0, Miami, FL). A low pulsatility peristaltic pump pushed HEPES-buffered solution (mM: 125 NaCl, 5.9 KCl, 2.56 CaCl2, 1 MgCl2, 25 HEPES, and 0.1% BSA; pH 7.4; and a perifusion rate of 100 μL/min) through a column containing 100 pancreatic islets immobilized in Bio-Gel P-4 Gel (BioRad, Hercules, CA). Glucose concentration was increased stepwise from 1, 3, 5, 7, to 11 mM (10 minutes each) with or without the glucagon receptor antagonists L-168,49 (50 nM) or des-His1-[Glu9]-Glucagon (1-29) amide (1μM). The perifusate was collected in an automatic fraction collector designed for a 96 well plate format. The columns containing the islets and the perifusion solutions were kept at 37°C, and the perifusate in the collecting plate was kept at < 4°C. Perifusates were collected every minute. Human insulin and mouse insulin release was determined in the perifusate with ELISAs (Mercodia).
Bio gel p 4 gel
Manufactured by Bio-Rad
Sourced in United States
Bio-Gel P-4 Gel is a size exclusion chromatography media used for the separation and purification of molecules based on their size and molecular weight. It is composed of cross-linked polyacrylamide beads that provide a porous structure for the separation process. The gel is suitable for the fractionation of proteins, peptides, and other biological macromolecules.
Automatically generated - may contain errors
Lab products found in correlation
Elisas, by Mercodia (1 mentions)
Ltq orbitrap velos, by Thermo Fisher Scientific (1 mentions)
Ultrasensitive mouse insulin elisa kit, by Mercodia (1 mentions)
Ultra low attachment plate, by Corning (1 mentions)
Model 680 microplate reader, by Bio-Rad (1 mentions)
Enzyme linked immunosorbent assay kit, by Mercodia (1 mentions)
8 protocols using bio gel p 4 gel
1
Dynamic Measurement of Islet Hormone Secretion
2
Dynamic Stimulation of Cell Secretion
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A high-capacity, automated perifusion system (BioRep® Perifusion
V2.0.0) was used to dynamically stimulate cell secretion. A low pulsatility
peristaltic pump was used to push HEPES-buffered solution (125 mM NaCl, 5.9 mM
KCl, 2.56 mM CaCl2, 1 mM MgCl2, 25 mM HEPES, 0.1% BSA, pH
7.4) through a sample container harboring 50 iPSC clusters or 20 HI immobilized
in Bio-Gel P-4 Gel (BioRad), or 700,000 EndoC-βH1 cells. Cells were stabilized
with a slow-flow perifusion rate (30 µl/min) with low glucose (2 mM) for 60
minutes. A combined stimulus (11 mM glucose with or without
3-isobutyl-1-methylxanthine - IBMX) was then added for 20 minutes at a flow rate
of 100 µl/min. Following a third step with 2 mM glucose for 20 minutes, cells
were exposed to 30 mM KCl for 20 minutes and then to 2 mM glucose for another 20
minutes. The perifusates were collected every minute by an automated fraction
collector designed for a multiwell plate format. Cells and perfusion solutions
were kept at 37°C in a built-in temperature controlled chamber, and collected
perifusates were stored at -20°C.
V2.0.0) was used to dynamically stimulate cell secretion. A low pulsatility
peristaltic pump was used to push HEPES-buffered solution (125 mM NaCl, 5.9 mM
KCl, 2.56 mM CaCl2, 1 mM MgCl2, 25 mM HEPES, 0.1% BSA, pH
7.4) through a sample container harboring 50 iPSC clusters or 20 HI immobilized
in Bio-Gel P-4 Gel (BioRad), or 700,000 EndoC-βH1 cells. Cells were stabilized
with a slow-flow perifusion rate (30 µl/min) with low glucose (2 mM) for 60
minutes. A combined stimulus (11 mM glucose with or without
3-isobutyl-1-methylxanthine - IBMX) was then added for 20 minutes at a flow rate
of 100 µl/min. Following a third step with 2 mM glucose for 20 minutes, cells
were exposed to 30 mM KCl for 20 minutes and then to 2 mM glucose for another 20
minutes. The perifusates were collected every minute by an automated fraction
collector designed for a multiwell plate format. Cells and perfusion solutions
were kept at 37°C in a built-in temperature controlled chamber, and collected
perifusates were stored at -20°C.
3
Purification of RNase B Glycans
4
Dynamic Insulin Secretion in Mouse Islets
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Mouse pancreatic islets were isolated by using a published protocol63 (link). An automated perifusion system was utilized to dynamically measure insulin secretion (Biorep Perifusion System, Miami, FL). A peristaltic pump pushed a HEPES-buffered solution (composition in mM: 125 NaCl, 5.9 KCl, 2.56 CaCl2, 1.2 MgCl2, 25 HEPES, and 0.1% BSA [pH 7.4]; perifusion rate: 100 μl/min) through a sample container with the islets immobilized in Bio-Gel P-4 Gel (Bio-Rad, Hercules, CA). Islets were preincubated with 3 mM glucose for 1 h and then incubated with 16 mM glucose for 15 min, followed by a final 20 min incubation step with 3 mM glucose. Glucose was applied with the HEPES-buffered solution, and the perfusate was collected every minute in a 96-well plate for further analysis. The chambers containing the islets were kept at 37 °C, and the perfusate in the collecting plate was kept at <4 °C. Insulin concentrations in the perfusates were determined with an ultrasensitive mouse insulin ELISA kit (Mercodia, Winston Salem, NC).
5
Dynamic Measurement of Islet Insulin Secretion
6
Dynamic Insulin Secretion Assay
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At the iβ step, an automated perifusion system (BioRep® Perifusion V2.0.0, Biorep Technologies Inc., Miami Lakes, FL, USA) was used to stimulate dynamically insulin secretion. The day before the test, cells were transferred in an ultra-low attachment plate (Costar) to allow cluster formation in suspension and BRE added at 50 μg/mL for 24 h. One thousand clusters (corresponding to approximately one million cells) were loaded in perifusion chambers and immobilized in Bio-Gel P-4 Gel (BioRad, Hercules, CA, USA). A peristaltic pump allows for the perfusion of cells with different solutions with a constant flow rate and a steady temperature of 37 °C, as previously described [11 (link),12 (link)]. Insulin released during dynamic perifusion was measured with the ELISA kit for human insulin (Mercodia, Winston Salem, NC, USA), following the manufacturer’s instructions. The plates were analyzed using an ELISA reader (MicroPlate Reader, Model 680, BioRad).
7
Dynamic Measurement of Islet Insulin Secretion
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A high-capacity, automated perifusion system was used to dynamically
measure insulin secretion from pancreatic islets (Biorep Perifusion V2.0.0,
Miami, FL). A low pulsatility peristaltic pump pushed KRBH solution at a
perifusion rate of 100 μL/min through a column containing 100 pancreatic
islets immobilized in Bio-Gel P-4 Gel (BioRad, Hercules, CA). Except otherwise
stated, glucose concentration was adjusted to 3 mM for all experiments. Stimuli
were applied with the perifusion buffer. The perifusate was collected in an
automatic fraction collector designed for a 96 well plate format. The columns
containing the islets and the perifusion solutions were kept at 37°C, and
the perifusate in the collecting plate was kept at < 4°C.
Perifusates were collected every minute. Insulin release in the perifusate was
determined with the human or mouse Mercodia Insulin ELISA kit following
manufacturer’s instructions.
measure insulin secretion from pancreatic islets (Biorep Perifusion V2.0.0,
Miami, FL). A low pulsatility peristaltic pump pushed KRBH solution at a
perifusion rate of 100 μL/min through a column containing 100 pancreatic
islets immobilized in Bio-Gel P-4 Gel (BioRad, Hercules, CA). Except otherwise
stated, glucose concentration was adjusted to 3 mM for all experiments. Stimuli
were applied with the perifusion buffer. The perifusate was collected in an
automatic fraction collector designed for a 96 well plate format. The columns
containing the islets and the perifusion solutions were kept at 37°C, and
the perifusate in the collecting plate was kept at < 4°C.
Perifusates were collected every minute. Insulin release in the perifusate was
determined with the human or mouse Mercodia Insulin ELISA kit following
manufacturer’s instructions.
8
Automated Perifusion System for Dynamic Cell Secretion
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A high-capacity automated perifusion system (BioRep Perifusion V2.0.0) was used to dynamically stimulate cell secretion. A low-pulsatility peristaltic pump was used to push 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-buffered solution (125 mM sodium chloride, 5.9 mM potassium chloride [KCl], 2.56 mM calcium chloride, 1 mM magnesium chloride, 25 mM 4-[2-hydroxyethyl]-1-piperazineethanesulfonic acid, 0.1% BSA, pH 7.4) through a sample container harboring 50 iPSC clusters in Bio-Gel P-4 gel (Bio-Rad). Cells were stabilized with a slow flow perifusion rate (30 mL/min) with low-glucose solution (2 mM) for 60 min, followed by 20 min at 100 mL/min. A combined stimulus (11 mM glucose with or without 50 mM 3-isobutyl-1-methylxanthine) was then added for 20 min. Following a third step with 2 mM glucose for 20 min, cells were exposed to 30 mM KCl for 20 min and then to 2 mM glucose for another 20 min. The perifusates were collected every minute and stored at À20°C until insulin dosage by enzyme-linked immunosorbent assay kit (Mercodia).
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