To measure the expression level of GLP-1R in vivo, a saturating dose of 1.2 nmol of AF647 monomer was injected in the tail vein of transgenic mice containing GFP under the control of the mouse insulin promoter (MIP-GFP mice, Jackson Laboratory Strain B6.Cg-Tg(Ins1-EGFP)1Hara/J). At 30 min post-injection, the mice were sacrificed and the pancreas resected. Successful islet targeting was confirmed using an Odyssey CLx scanner and by verifying colocalization of GFP and AF647 signal using fluorescence microscopy. After confirming specific islet targeting, the pancreas was incubated at 37°C for 15 min in 1 mg/mL collagenase type XI in PBS with continuous mixing to obtain a single cell suspension. The cells were analyzed with an Attune Acoustic Focusing Cytometer to determine the fluorescence intensity of AF647 on the GFP-positive β-cells. After accounting for quenching effects of conjugating AF647 to exendin, a calibration curve generated with Quantum Alexa Fluor 647 MESF beads (Bangs Laboratories, Inc.) was used to determine the GLP-1R expression level in vivo.
Quantum alexa fluor 647 mesf beads
Manufactured by Bangs Laboratories
Quantum Alexa Fluor 647 MESF beads are fluorescent calibration beads designed for the quantification of fluorescence intensity. The beads are composed of polystyrene and contain the Alexa Fluor 647 fluorophore. They provide a standardized fluorescence signal that can be used to determine the Molecules of Equivalent Soluble Fluorophore (MESF) of samples.
Automatically generated - may contain errors
2 protocols using quantum alexa fluor 647 mesf beads
1
Quantifying GLP-1R Expression in Pancreatic Islets
2
Quantifying Cellular Uptake of siRNA
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A431-d2EGFP cells were seeded at a density of 15 000 cells/well in 96-well plates 16–20 h prior to the experiment. The p19-E18 constructs were incubated with fluorescently labeled siRNA (Seq I) at a 1:1 molar ratio for 30 min at 4°C. The p19-E18/siRNA complexes were then diluted in DMEM containing 10% FBS at varying concentrations and incubated with cells for 0–6 h in a reverse timecourse. For competition experiments, incubation was performed with 20 nM of p19-E18/siRNA complexes and varying concentrations (0–4 μM) of SUMO-E18 in complete media. After 6 h, cells were washed with PBS, trypsinized, neutralized with cold PBSA containing 2% FBS and analyzed on an iQue Screener (IntelliCyt). All liquid handling was performed using an EL406 plate washer (BioTek) and a Freedom EVO 150 liquid handling system (Tecan) to minimize variability. Background from untreated cells were subtracted from all measurements, which were then converted to number of fluorophores using Quantum Alexa Fluor 647 MESF beads following manufacturer's instructions (Bangs Laboratories).
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