downward red dag sensor  (Montana Molecular)

Journal: Diabetes

Article Title: Nicotinic Signaling Stimulates Glucagon Secretion in Mouse and Human Pancreatic α-Cells

doi: 10.2337/db23-0809

Figure Lengend Snippet: ACh elevates [DAG] i in α- and β-cells. A : Heat map outlining dynamics of [DAG] i in mouse islet α- and β-cells, in response to 10 μmol/L ACh, imaged using red fluorescent recombinant sensor Downward DAG. α- and β-cells were distinguished according to their positive and negative [cAMP] i response to 10 μmol/L adrenaline, which was imaged concurrently via green reporter of [cAMP] i , Upward cADDis. B : Quantification of [DAG] i data ( A ) for α- ( n = 51; five experiments) and β-cells ( n = 57). C : Representative traces of Ca 2+ dynamics evoked by ACh in pancreatic islet α-cells in presence of 5 mmol/L Ca 2+ chelator EGTA. D : Quantification of [Ca 2+ ] i data ( n = 22; five experiments) ( C ). E and F : Representative current-clamp recordings of plasma membrane electrical potential in α-cells in presence of 10 μmol/L ACh ( n = 6) ( E ) or 10 μmol/L nicotine ( n = 12) ( F ); arrow ( E ) indicates transient repolarization. G : Quantification of V m data ( n = 18, n = 6, n = 12) ( E and F ) at level of action potential amplitude and frequency. #Differences vs. 3 mmol/L glucose alone and *3 mmol/L glucose + 10 μmol/L ACh in α-cells are significant ( P < 0.05). pAUC, partial area under curve.

Article Snippet: Recombinant sensors (Downward Green DAG [diacylglycerol], Upward Red cADDis) were delivered via BacMam (Montana Molecular, Bozeman, MT) vectors at 10 5 international units per islet, followed by 36-h culturing (as above) to express the proteins.

Techniques: Recombinant, Membrane

Journal: bioRxiv

Article Title: A PACAP-activated network for secretion requires coordination of Ca 2+ influx and Ca 2+ mobilization

doi: 10.1101/2024.01.03.574069

Figure Lengend Snippet: A. PACAP stimulation caused increases in cADDis (cAMP indicator) and R-GECO (Ca 2+ indicator) fluorescence (n = 5). B . cADDis and R-GECO intensity versus time records from cell in A . C . Three representative records showing that PLCε KO inhibited PACAP-evoked Ca 2+ transients but not cAMP production (n=5). D . cAMP production (measured via cADDiS fluorescence) is normal in PLCε KO cells stimulated by PACAP with or without extracellular Ca 2+ . The three curves are essentially overlapping (student’s t-test; p > 0.05). Individual data points on curves represented as means ± SEM. n=13 for PACAP+Ca 2+ stimulation of WT cells; n=24 for PACAP+Ca 2+ stimulation of PLCε KO cells; n=11 for PACAP-Ca 2+ stimulation of WT cells. E . Examples of WT (n=26) and KO (n=26) cells expressing a downward red DAG sensor (Montana Molecular) stimulated by PACAP (n=26). F. DAG fluorescence decreased in WT cells after PACAP application, but not in KO cells. Differences between data sets were statistically significant (p < 0.05) after 30 s.

Article Snippet: Cells were transduced with either green cADDis (cAMP) upward sensor (Montana Molecular, Cat# U0200G), red GECO Ca 2+ upward sensor (Montana Molecular, Cat# U-0600R), or red DAG downward sensor (Montana Molecular, Cat# D-0300R) following the manufacturer’s protocol.

Techniques: Fluorescence, Expressing