Structured Review

Dojindo Labs fura2 am
Effect of ibuprofen or sodium valproate on [Ca 2+ ] i induced by thrombin. Notes: <t>Fura2-loaded</t> untreated HEL cells and PMA-induced HEL cells were treated with ibuprofen ( A ; 0.8–200 µM) or sodium valproate ( B ; 50–1,000 µg/mL) for 6 minutes, then stimulated with thrombin. ■, untreated HEL cells; □, PMA-induced HEL cells. Intracellular Ca 2+ mobilization was induced by thrombin. Results are presented as the mean ± SD, n=5. ** P
Fura2 Am, supplied by Dojindo Labs, used in various techniques. Bioz Stars score: 92/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/fura2 am/product/Dojindo Labs
Average 92 stars, based on 9 article reviews
Price from $9.99 to $1999.99
fura2 am - by Bioz Stars, 2020-09
92/100 stars

Images

1) Product Images from "Evaluation of effects of various drugs on platelet functions using phorbol 12-myristate 13-acetate-induced megakaryocytic human erythroid leukemia cells"

Article Title: Evaluation of effects of various drugs on platelet functions using phorbol 12-myristate 13-acetate-induced megakaryocytic human erythroid leukemia cells

Journal: Drug Design, Development and Therapy

doi: 10.2147/DDDT.S115910

Effect of ibuprofen or sodium valproate on [Ca 2+ ] i induced by thrombin. Notes: Fura2-loaded untreated HEL cells and PMA-induced HEL cells were treated with ibuprofen ( A ; 0.8–200 µM) or sodium valproate ( B ; 50–1,000 µg/mL) for 6 minutes, then stimulated with thrombin. ■, untreated HEL cells; □, PMA-induced HEL cells. Intracellular Ca 2+ mobilization was induced by thrombin. Results are presented as the mean ± SD, n=5. ** P
Figure Legend Snippet: Effect of ibuprofen or sodium valproate on [Ca 2+ ] i induced by thrombin. Notes: Fura2-loaded untreated HEL cells and PMA-induced HEL cells were treated with ibuprofen ( A ; 0.8–200 µM) or sodium valproate ( B ; 50–1,000 µg/mL) for 6 minutes, then stimulated with thrombin. ■, untreated HEL cells; □, PMA-induced HEL cells. Intracellular Ca 2+ mobilization was induced by thrombin. Results are presented as the mean ± SD, n=5. ** P

Techniques Used:

Effect of aspirin or cilostazol on [Ca 2+ ] i induced by thrombin. Notes: Fura2-loaded untreated HEL cells and PMA-induced HEL cells were treated with aspirin ( A ; 5.6–560 µM) or cilostazol ( B ; 1–10 µM) for 6 minutes and then stimulated with thrombin. ■, untreated HEL cells; □, PMA-induced HEL cells. Intracellular Ca 2+ mobilization was induced by thrombin. Results are presented as the mean ± SD, n=5. ** P
Figure Legend Snippet: Effect of aspirin or cilostazol on [Ca 2+ ] i induced by thrombin. Notes: Fura2-loaded untreated HEL cells and PMA-induced HEL cells were treated with aspirin ( A ; 5.6–560 µM) or cilostazol ( B ; 1–10 µM) for 6 minutes and then stimulated with thrombin. ■, untreated HEL cells; □, PMA-induced HEL cells. Intracellular Ca 2+ mobilization was induced by thrombin. Results are presented as the mean ± SD, n=5. ** P

Techniques Used:

Effect of drug combinations on [Ca 2+ ] i induced by thrombin. Notes: Fura2-loaded PMA-induced HEL cells were treated with the simultaneous combined use of aspirin (ASA) and ibuprofen (IBU) ( A and B ) or sodium valproate (VPA) ( C ) for 6 minutes, then stimulated with thrombin. Intracellular Ca 2+ mobilization was induced by thrombin. Results are presented as the mean ± SD, n=5. ** P
Figure Legend Snippet: Effect of drug combinations on [Ca 2+ ] i induced by thrombin. Notes: Fura2-loaded PMA-induced HEL cells were treated with the simultaneous combined use of aspirin (ASA) and ibuprofen (IBU) ( A and B ) or sodium valproate (VPA) ( C ) for 6 minutes, then stimulated with thrombin. Intracellular Ca 2+ mobilization was induced by thrombin. Results are presented as the mean ± SD, n=5. ** P

Techniques Used:

2) Product Images from "Cell surface flip-flop of phosphatidylserine is critical for PIEZO1-mediated myotube formation"

Article Title: Cell surface flip-flop of phosphatidylserine is critical for PIEZO1-mediated myotube formation

Journal: Nature Communications

doi: 10.1038/s41467-018-04436-w

Impaired PIEZO1 activation in PS-exposing human primary myoblasts during myotube formation. a Detection of cell surface-exposed PS on differentiating human primary myoblasts by annexin V-GFP (PS, green), DAPI (nuclei, blue) and fixable viability dye (dead cells, red). b Fura2 imaging of Ca 2+ influx in annexin V-GFP-labelled human primary myoblasts (arrow) upon addition of Yoda1 and ionomycin. Representative traces ( c ), quantification of Yoda1-induced Ca 2+ influx ( d ) and basal Ca 2+ level ( e ) in b . **** P
Figure Legend Snippet: Impaired PIEZO1 activation in PS-exposing human primary myoblasts during myotube formation. a Detection of cell surface-exposed PS on differentiating human primary myoblasts by annexin V-GFP (PS, green), DAPI (nuclei, blue) and fixable viability dye (dead cells, red). b Fura2 imaging of Ca 2+ influx in annexin V-GFP-labelled human primary myoblasts (arrow) upon addition of Yoda1 and ionomycin. Representative traces ( c ), quantification of Yoda1-induced Ca 2+ influx ( d ) and basal Ca 2+ level ( e ) in b . **** P

Techniques Used: Activation Assay, Imaging

Impaired PIEZO1 activation in PS flippase-deficient myoblasts. a , b Suppression of agonist-induced PIEZO1 activation by PS flippase deficiency. a Left: Fura2 ratiometric measurements (F 340 /F 380 ) of Yoda1-induced Ca 2+ influx in WT, PIEZO1-deficient, CDC50A-deficient, and ATP11A-deficient C2C12 myoblasts. Right: quantification of Yoda1-induced Ca 2+ influx as the maximal increment of F 340 /F 380 (∆ ratio) in left. b Left: Fura2 ratiometric measurements of Yoda1-induced Ca 2+ influx in human primary myoblasts transfected with control, PIEZO1, CDC50A or ATP11A siRNA. Right: quantification of Yoda1-induced Ca 2+ influx in left. c Rescue of agonist-induced PIEZO1 activation in PS flippase-deficient myoblasts by overexpression of PS flippase complex components. Quantification of Yoda1-induced Ca 2+ influx in CDC50A-deficient (left) or ATP11A-deficient (right) C2C12 myoblasts expressing FLAG-tagged CDC50A, ATP11A, ATP11B or ATP11C. d , e Normal plasma membrane tension in PS flippase-deficient myoblasts. d Schematic model of membrane tension measurement using an optical trap. e Quantification of membrane tension in WT, CDC50A-deficient and cytochalasin D-treated WT C2C12 cells. f Schematic model showing PS flippase-mediated inward translocation of cell surface-exposed PS as a prerequisite for PIEZO1 activation. ** P
Figure Legend Snippet: Impaired PIEZO1 activation in PS flippase-deficient myoblasts. a , b Suppression of agonist-induced PIEZO1 activation by PS flippase deficiency. a Left: Fura2 ratiometric measurements (F 340 /F 380 ) of Yoda1-induced Ca 2+ influx in WT, PIEZO1-deficient, CDC50A-deficient, and ATP11A-deficient C2C12 myoblasts. Right: quantification of Yoda1-induced Ca 2+ influx as the maximal increment of F 340 /F 380 (∆ ratio) in left. b Left: Fura2 ratiometric measurements of Yoda1-induced Ca 2+ influx in human primary myoblasts transfected with control, PIEZO1, CDC50A or ATP11A siRNA. Right: quantification of Yoda1-induced Ca 2+ influx in left. c Rescue of agonist-induced PIEZO1 activation in PS flippase-deficient myoblasts by overexpression of PS flippase complex components. Quantification of Yoda1-induced Ca 2+ influx in CDC50A-deficient (left) or ATP11A-deficient (right) C2C12 myoblasts expressing FLAG-tagged CDC50A, ATP11A, ATP11B or ATP11C. d , e Normal plasma membrane tension in PS flippase-deficient myoblasts. d Schematic model of membrane tension measurement using an optical trap. e Quantification of membrane tension in WT, CDC50A-deficient and cytochalasin D-treated WT C2C12 cells. f Schematic model showing PS flippase-mediated inward translocation of cell surface-exposed PS as a prerequisite for PIEZO1 activation. ** P

Techniques Used: Activation Assay, Transfection, Over Expression, Expressing, Translocation Assay

3) Product Images from "Interactions between ?CaMKII and calmodulin in living cells: conformational changes arising from CaM -dependent and -independent relationships"

Article Title: Interactions between ?CaMKII and calmodulin in living cells: conformational changes arising from CaM -dependent and -independent relationships

Journal: Molecular Brain

doi: 10.1186/1756-6606-6-37

Time lapse imaging of αCaMKII activation in HeLa cells. (A) Changes in intracellular Ca 2+ concentration measured using Fura2-AM. Upper panels show images of typical 340 nm/380 nm ratio at each time point before and after ionomycin application. Lower graph shows changes in relative 340 nm/380 nm ratio (open circle, ionomycin; filled circle, DMSO). Black bar shows the period of ionomycin application. 12–18 cells were analyzed in each group. (B, C) Changes in the interaction of YFP-αCaMKII with CFP-CaM (B) and the conformational change of YFP-αCaMKII-CFP (C) . Upper panels show images of typical YFP/CFP emission ratio before and after ionomycin application (upper images, –KN-93; lower images, +KN-93). Lower graphs show changes in relative YFP/CFP emission ratio (open circle, ionomycin; filled circle, ionomycin with KN-93). Black bar shows the period of ionomycin application. 9–13 cells were analyzed in each group. (D) Effect of removal of ionomycin by wash out at 0.5 or 1 min following ionomycin application. Upper graph shows the interaction of YFP-αCaMKII with CFP-CaM (open circle, –wash; triangle, 0.5 min; filled circle, 1 min). Lower graph shows the conformational change of YFP-αCaMKII-CFP (open circle, -wash; triangle, 0.5 min; filled circle, 1 min). 10–12 cells were analyzed in each group. (E) Auto-phosphorylation at T286 of αCaMKII fusion proteins after ionomycin application at each time point (0, 1, 5 and 10 min). Upper panel shows Western blot analyses analyzing the expression level of phospho-αCaMKII fusion proteins and total-αCaMKII fusion proteins. Lower graph shows relative ratio of phospho-αCaMKII/αCaMKII levels (n = 3).
Figure Legend Snippet: Time lapse imaging of αCaMKII activation in HeLa cells. (A) Changes in intracellular Ca 2+ concentration measured using Fura2-AM. Upper panels show images of typical 340 nm/380 nm ratio at each time point before and after ionomycin application. Lower graph shows changes in relative 340 nm/380 nm ratio (open circle, ionomycin; filled circle, DMSO). Black bar shows the period of ionomycin application. 12–18 cells were analyzed in each group. (B, C) Changes in the interaction of YFP-αCaMKII with CFP-CaM (B) and the conformational change of YFP-αCaMKII-CFP (C) . Upper panels show images of typical YFP/CFP emission ratio before and after ionomycin application (upper images, –KN-93; lower images, +KN-93). Lower graphs show changes in relative YFP/CFP emission ratio (open circle, ionomycin; filled circle, ionomycin with KN-93). Black bar shows the period of ionomycin application. 9–13 cells were analyzed in each group. (D) Effect of removal of ionomycin by wash out at 0.5 or 1 min following ionomycin application. Upper graph shows the interaction of YFP-αCaMKII with CFP-CaM (open circle, –wash; triangle, 0.5 min; filled circle, 1 min). Lower graph shows the conformational change of YFP-αCaMKII-CFP (open circle, -wash; triangle, 0.5 min; filled circle, 1 min). 10–12 cells were analyzed in each group. (E) Auto-phosphorylation at T286 of αCaMKII fusion proteins after ionomycin application at each time point (0, 1, 5 and 10 min). Upper panel shows Western blot analyses analyzing the expression level of phospho-αCaMKII fusion proteins and total-αCaMKII fusion proteins. Lower graph shows relative ratio of phospho-αCaMKII/αCaMKII levels (n = 3).

Techniques Used: Imaging, Activation Assay, Concentration Assay, Chick Chorioallantoic Membrane Assay, Western Blot, Expressing

Time lapse imaging of αCaMKII activation in SH-SY5Y cells. (A) Changes in intracellular Ca 2+ concentration measured using Fura2-AM. Upper panels show images of typical 340 nm/380 nm ratio at each time point before and after the application of ionomycin or KCl (50 mM)-application. Lower graph shows changes in relative 340 nm/380 nm ratio (open circle, ionomycin; filled circle, KCl). Black bar shows the period of ionomycin or KCl application. 30–40 cells were analyzed in each group. (B, C) Changes in the interaction of YFP-αCaMKII with CFP-CaM (B) and the conformational change of YFP-αCaMKII-CFP (C) . Upper panels show images of typical YFP/CFP emission ratio before and after ionomycin or KCl application. Lower graphs show changes in relative YFP/CFP emission ratio (open circle, ionomycin; filled circle, KCl). Black bar shows the period of ionomycin or KCl application. 11–25 cells were analyzed in each group. (D) Auto-phosphorylation at T286 of αCaMKII fusion proteins after ionomycin or KCI application at each time point (0, 1, 5 and 10 min). Upper panel shows Western blot analyses analyzing the expression level of phospho-αCaMKII fusion proteins and total-αCaMKII fusion proteins. Lower graph shows relative ratio of phospho-αCaMKII/αCaMKII levels (n = 3).
Figure Legend Snippet: Time lapse imaging of αCaMKII activation in SH-SY5Y cells. (A) Changes in intracellular Ca 2+ concentration measured using Fura2-AM. Upper panels show images of typical 340 nm/380 nm ratio at each time point before and after the application of ionomycin or KCl (50 mM)-application. Lower graph shows changes in relative 340 nm/380 nm ratio (open circle, ionomycin; filled circle, KCl). Black bar shows the period of ionomycin or KCl application. 30–40 cells were analyzed in each group. (B, C) Changes in the interaction of YFP-αCaMKII with CFP-CaM (B) and the conformational change of YFP-αCaMKII-CFP (C) . Upper panels show images of typical YFP/CFP emission ratio before and after ionomycin or KCl application. Lower graphs show changes in relative YFP/CFP emission ratio (open circle, ionomycin; filled circle, KCl). Black bar shows the period of ionomycin or KCl application. 11–25 cells were analyzed in each group. (D) Auto-phosphorylation at T286 of αCaMKII fusion proteins after ionomycin or KCI application at each time point (0, 1, 5 and 10 min). Upper panel shows Western blot analyses analyzing the expression level of phospho-αCaMKII fusion proteins and total-αCaMKII fusion proteins. Lower graph shows relative ratio of phospho-αCaMKII/αCaMKII levels (n = 3).

Techniques Used: Imaging, Activation Assay, Concentration Assay, Chick Chorioallantoic Membrane Assay, Western Blot, Expressing

4) Product Images from "Cell surface flip-flop of phosphatidylserine is critical for PIEZO1-mediated myotube formation"

Article Title: Cell surface flip-flop of phosphatidylserine is critical for PIEZO1-mediated myotube formation

Journal: Nature Communications

doi: 10.1038/s41467-018-04436-w

Impaired PIEZO1 activation in PS-exposing human primary myoblasts during myotube formation. a Detection of cell surface-exposed PS on differentiating human primary myoblasts by annexin V-GFP (PS, green), DAPI (nuclei, blue) and fixable viability dye (dead cells, red). b Fura2 imaging of Ca 2+ influx in annexin V-GFP-labelled human primary myoblasts (arrow) upon addition of Yoda1 and ionomycin. Representative traces ( c ), quantification of Yoda1-induced Ca 2+ influx ( d ) and basal Ca 2+ level ( e ) in b . **** P
Figure Legend Snippet: Impaired PIEZO1 activation in PS-exposing human primary myoblasts during myotube formation. a Detection of cell surface-exposed PS on differentiating human primary myoblasts by annexin V-GFP (PS, green), DAPI (nuclei, blue) and fixable viability dye (dead cells, red). b Fura2 imaging of Ca 2+ influx in annexin V-GFP-labelled human primary myoblasts (arrow) upon addition of Yoda1 and ionomycin. Representative traces ( c ), quantification of Yoda1-induced Ca 2+ influx ( d ) and basal Ca 2+ level ( e ) in b . **** P

Techniques Used: Activation Assay, Imaging

Impaired PIEZO1 activation in PS flippase-deficient myoblasts. a , b Suppression of agonist-induced PIEZO1 activation by PS flippase deficiency. a Left: Fura2 ratiometric measurements (F 340 /F 380 ) of Yoda1-induced Ca 2+ influx in WT, PIEZO1-deficient, CDC50A-deficient, and ATP11A-deficient C2C12 myoblasts. Right: quantification of Yoda1-induced Ca 2+ influx as the maximal increment of F 340 /F 380 (∆ ratio) in left. b Left: Fura2 ratiometric measurements of Yoda1-induced Ca 2+ influx in human primary myoblasts transfected with control, PIEZO1, CDC50A or ATP11A siRNA. Right: quantification of Yoda1-induced Ca 2+ influx in left. c Rescue of agonist-induced PIEZO1 activation in PS flippase-deficient myoblasts by overexpression of PS flippase complex components. Quantification of Yoda1-induced Ca 2+ influx in CDC50A-deficient (left) or ATP11A-deficient (right) C2C12 myoblasts expressing FLAG-tagged CDC50A, ATP11A, ATP11B or ATP11C. d , e Normal plasma membrane tension in PS flippase-deficient myoblasts. d Schematic model of membrane tension measurement using an optical trap. e Quantification of membrane tension in WT, CDC50A-deficient and cytochalasin D-treated WT C2C12 cells. f Schematic model showing PS flippase-mediated inward translocation of cell surface-exposed PS as a prerequisite for PIEZO1 activation. ** P
Figure Legend Snippet: Impaired PIEZO1 activation in PS flippase-deficient myoblasts. a , b Suppression of agonist-induced PIEZO1 activation by PS flippase deficiency. a Left: Fura2 ratiometric measurements (F 340 /F 380 ) of Yoda1-induced Ca 2+ influx in WT, PIEZO1-deficient, CDC50A-deficient, and ATP11A-deficient C2C12 myoblasts. Right: quantification of Yoda1-induced Ca 2+ influx as the maximal increment of F 340 /F 380 (∆ ratio) in left. b Left: Fura2 ratiometric measurements of Yoda1-induced Ca 2+ influx in human primary myoblasts transfected with control, PIEZO1, CDC50A or ATP11A siRNA. Right: quantification of Yoda1-induced Ca 2+ influx in left. c Rescue of agonist-induced PIEZO1 activation in PS flippase-deficient myoblasts by overexpression of PS flippase complex components. Quantification of Yoda1-induced Ca 2+ influx in CDC50A-deficient (left) or ATP11A-deficient (right) C2C12 myoblasts expressing FLAG-tagged CDC50A, ATP11A, ATP11B or ATP11C. d , e Normal plasma membrane tension in PS flippase-deficient myoblasts. d Schematic model of membrane tension measurement using an optical trap. e Quantification of membrane tension in WT, CDC50A-deficient and cytochalasin D-treated WT C2C12 cells. f Schematic model showing PS flippase-mediated inward translocation of cell surface-exposed PS as a prerequisite for PIEZO1 activation. ** P

Techniques Used: Activation Assay, Transfection, Over Expression, Expressing, Translocation Assay

5) Product Images from "Cell surface flip-flop of phosphatidylserine is critical for PIEZO1-mediated myotube formation"

Article Title: Cell surface flip-flop of phosphatidylserine is critical for PIEZO1-mediated myotube formation

Journal: Nature Communications

doi: 10.1038/s41467-018-04436-w

Impaired PIEZO1 activation in PS-exposing human primary myoblasts during myotube formation. a Detection of cell surface-exposed PS on differentiating human primary myoblasts by annexin V-GFP (PS, green), DAPI (nuclei, blue) and fixable viability dye (dead cells, red). b Fura2 imaging of Ca 2+ influx in annexin V-GFP-labelled human primary myoblasts (arrow) upon addition of Yoda1 and ionomycin. Representative traces ( c ), quantification of Yoda1-induced Ca 2+ influx ( d ) and basal Ca 2+ level ( e ) in b . **** P
Figure Legend Snippet: Impaired PIEZO1 activation in PS-exposing human primary myoblasts during myotube formation. a Detection of cell surface-exposed PS on differentiating human primary myoblasts by annexin V-GFP (PS, green), DAPI (nuclei, blue) and fixable viability dye (dead cells, red). b Fura2 imaging of Ca 2+ influx in annexin V-GFP-labelled human primary myoblasts (arrow) upon addition of Yoda1 and ionomycin. Representative traces ( c ), quantification of Yoda1-induced Ca 2+ influx ( d ) and basal Ca 2+ level ( e ) in b . **** P

Techniques Used: Activation Assay, Imaging

Impaired PIEZO1 activation in PS flippase-deficient myoblasts. a , b Suppression of agonist-induced PIEZO1 activation by PS flippase deficiency. a Left: Fura2 ratiometric measurements (F 340 /F 380 ) of Yoda1-induced Ca 2+ influx in WT, PIEZO1-deficient, CDC50A-deficient, and ATP11A-deficient C2C12 myoblasts. Right: quantification of Yoda1-induced Ca 2+ influx as the maximal increment of F 340 /F 380 (∆ ratio) in left. b Left: Fura2 ratiometric measurements of Yoda1-induced Ca 2+ influx in human primary myoblasts transfected with control, PIEZO1, CDC50A or ATP11A siRNA. Right: quantification of Yoda1-induced Ca 2+ influx in left. c Rescue of agonist-induced PIEZO1 activation in PS flippase-deficient myoblasts by overexpression of PS flippase complex components. Quantification of Yoda1-induced Ca 2+ influx in CDC50A-deficient (left) or ATP11A-deficient (right) C2C12 myoblasts expressing FLAG-tagged CDC50A, ATP11A, ATP11B or ATP11C. d , e Normal plasma membrane tension in PS flippase-deficient myoblasts. d Schematic model of membrane tension measurement using an optical trap. e Quantification of membrane tension in WT, CDC50A-deficient and cytochalasin D-treated WT C2C12 cells. f Schematic model showing PS flippase-mediated inward translocation of cell surface-exposed PS as a prerequisite for PIEZO1 activation. ** P
Figure Legend Snippet: Impaired PIEZO1 activation in PS flippase-deficient myoblasts. a , b Suppression of agonist-induced PIEZO1 activation by PS flippase deficiency. a Left: Fura2 ratiometric measurements (F 340 /F 380 ) of Yoda1-induced Ca 2+ influx in WT, PIEZO1-deficient, CDC50A-deficient, and ATP11A-deficient C2C12 myoblasts. Right: quantification of Yoda1-induced Ca 2+ influx as the maximal increment of F 340 /F 380 (∆ ratio) in left. b Left: Fura2 ratiometric measurements of Yoda1-induced Ca 2+ influx in human primary myoblasts transfected with control, PIEZO1, CDC50A or ATP11A siRNA. Right: quantification of Yoda1-induced Ca 2+ influx in left. c Rescue of agonist-induced PIEZO1 activation in PS flippase-deficient myoblasts by overexpression of PS flippase complex components. Quantification of Yoda1-induced Ca 2+ influx in CDC50A-deficient (left) or ATP11A-deficient (right) C2C12 myoblasts expressing FLAG-tagged CDC50A, ATP11A, ATP11B or ATP11C. d , e Normal plasma membrane tension in PS flippase-deficient myoblasts. d Schematic model of membrane tension measurement using an optical trap. e Quantification of membrane tension in WT, CDC50A-deficient and cytochalasin D-treated WT C2C12 cells. f Schematic model showing PS flippase-mediated inward translocation of cell surface-exposed PS as a prerequisite for PIEZO1 activation. ** P

Techniques Used: Activation Assay, Transfection, Over Expression, Expressing, Translocation Assay

6) Product Images from "Interactions between ?CaMKII and calmodulin in living cells: conformational changes arising from CaM -dependent and -independent relationships"

Article Title: Interactions between ?CaMKII and calmodulin in living cells: conformational changes arising from CaM -dependent and -independent relationships

Journal: Molecular Brain

doi: 10.1186/1756-6606-6-37

Time lapse imaging of αCaMKII activation in HeLa cells. (A) Changes in intracellular Ca 2+ concentration measured using Fura2-AM. Upper panels show images of typical 340 nm/380 nm ratio at each time point before and after ionomycin application. Lower graph shows changes in relative 340 nm/380 nm ratio (open circle, ionomycin; filled circle, DMSO). Black bar shows the period of ionomycin application. 12–18 cells were analyzed in each group. (B, C) Changes in the interaction of YFP-αCaMKII with CFP-CaM (B) and the conformational change of YFP-αCaMKII-CFP (C) . Upper panels show images of typical YFP/CFP emission ratio before and after ionomycin application (upper images, –KN-93; lower images, +KN-93). Lower graphs show changes in relative YFP/CFP emission ratio (open circle, ionomycin; filled circle, ionomycin with KN-93). Black bar shows the period of ionomycin application. 9–13 cells were analyzed in each group. (D) Effect of removal of ionomycin by wash out at 0.5 or 1 min following ionomycin application. Upper graph shows the interaction of YFP-αCaMKII with CFP-CaM (open circle, –wash; triangle, 0.5 min; filled circle, 1 min). Lower graph shows the conformational change of YFP-αCaMKII-CFP (open circle, -wash; triangle, 0.5 min; filled circle, 1 min). 10–12 cells were analyzed in each group. (E) Auto-phosphorylation at T286 of αCaMKII fusion proteins after ionomycin application at each time point (0, 1, 5 and 10 min). Upper panel shows Western blot analyses analyzing the expression level of phospho-αCaMKII fusion proteins and total-αCaMKII fusion proteins. Lower graph shows relative ratio of phospho-αCaMKII/αCaMKII levels (n = 3).
Figure Legend Snippet: Time lapse imaging of αCaMKII activation in HeLa cells. (A) Changes in intracellular Ca 2+ concentration measured using Fura2-AM. Upper panels show images of typical 340 nm/380 nm ratio at each time point before and after ionomycin application. Lower graph shows changes in relative 340 nm/380 nm ratio (open circle, ionomycin; filled circle, DMSO). Black bar shows the period of ionomycin application. 12–18 cells were analyzed in each group. (B, C) Changes in the interaction of YFP-αCaMKII with CFP-CaM (B) and the conformational change of YFP-αCaMKII-CFP (C) . Upper panels show images of typical YFP/CFP emission ratio before and after ionomycin application (upper images, –KN-93; lower images, +KN-93). Lower graphs show changes in relative YFP/CFP emission ratio (open circle, ionomycin; filled circle, ionomycin with KN-93). Black bar shows the period of ionomycin application. 9–13 cells were analyzed in each group. (D) Effect of removal of ionomycin by wash out at 0.5 or 1 min following ionomycin application. Upper graph shows the interaction of YFP-αCaMKII with CFP-CaM (open circle, –wash; triangle, 0.5 min; filled circle, 1 min). Lower graph shows the conformational change of YFP-αCaMKII-CFP (open circle, -wash; triangle, 0.5 min; filled circle, 1 min). 10–12 cells were analyzed in each group. (E) Auto-phosphorylation at T286 of αCaMKII fusion proteins after ionomycin application at each time point (0, 1, 5 and 10 min). Upper panel shows Western blot analyses analyzing the expression level of phospho-αCaMKII fusion proteins and total-αCaMKII fusion proteins. Lower graph shows relative ratio of phospho-αCaMKII/αCaMKII levels (n = 3).

Techniques Used: Imaging, Activation Assay, Concentration Assay, Chick Chorioallantoic Membrane Assay, Western Blot, Expressing

Time lapse imaging of αCaMKII activation in SH-SY5Y cells. (A) Changes in intracellular Ca 2+ concentration measured using Fura2-AM. Upper panels show images of typical 340 nm/380 nm ratio at each time point before and after the application of ionomycin or KCl (50 mM)-application. Lower graph shows changes in relative 340 nm/380 nm ratio (open circle, ionomycin; filled circle, KCl). Black bar shows the period of ionomycin or KCl application. 30–40 cells were analyzed in each group. (B, C) Changes in the interaction of YFP-αCaMKII with CFP-CaM (B) and the conformational change of YFP-αCaMKII-CFP (C) . Upper panels show images of typical YFP/CFP emission ratio before and after ionomycin or KCl application. Lower graphs show changes in relative YFP/CFP emission ratio (open circle, ionomycin; filled circle, KCl). Black bar shows the period of ionomycin or KCl application. 11–25 cells were analyzed in each group. (D) Auto-phosphorylation at T286 of αCaMKII fusion proteins after ionomycin or KCI application at each time point (0, 1, 5 and 10 min). Upper panel shows Western blot analyses analyzing the expression level of phospho-αCaMKII fusion proteins and total-αCaMKII fusion proteins. Lower graph shows relative ratio of phospho-αCaMKII/αCaMKII levels (n = 3).
Figure Legend Snippet: Time lapse imaging of αCaMKII activation in SH-SY5Y cells. (A) Changes in intracellular Ca 2+ concentration measured using Fura2-AM. Upper panels show images of typical 340 nm/380 nm ratio at each time point before and after the application of ionomycin or KCl (50 mM)-application. Lower graph shows changes in relative 340 nm/380 nm ratio (open circle, ionomycin; filled circle, KCl). Black bar shows the period of ionomycin or KCl application. 30–40 cells were analyzed in each group. (B, C) Changes in the interaction of YFP-αCaMKII with CFP-CaM (B) and the conformational change of YFP-αCaMKII-CFP (C) . Upper panels show images of typical YFP/CFP emission ratio before and after ionomycin or KCl application. Lower graphs show changes in relative YFP/CFP emission ratio (open circle, ionomycin; filled circle, KCl). Black bar shows the period of ionomycin or KCl application. 11–25 cells were analyzed in each group. (D) Auto-phosphorylation at T286 of αCaMKII fusion proteins after ionomycin or KCI application at each time point (0, 1, 5 and 10 min). Upper panel shows Western blot analyses analyzing the expression level of phospho-αCaMKII fusion proteins and total-αCaMKII fusion proteins. Lower graph shows relative ratio of phospho-αCaMKII/αCaMKII levels (n = 3).

Techniques Used: Imaging, Activation Assay, Concentration Assay, Chick Chorioallantoic Membrane Assay, Western Blot, Expressing

7) Product Images from "Cell surface flip-flop of phosphatidylserine is critical for PIEZO1-mediated myotube formation"

Article Title: Cell surface flip-flop of phosphatidylserine is critical for PIEZO1-mediated myotube formation

Journal: Nature Communications

doi: 10.1038/s41467-018-04436-w

Impaired PIEZO1 activation in PS-exposing human primary myoblasts during myotube formation. a Detection of cell surface-exposed PS on differentiating human primary myoblasts by annexin V-GFP (PS, green), DAPI (nuclei, blue) and fixable viability dye (dead cells, red). b Fura2 imaging of Ca 2+ influx in annexin V-GFP-labelled human primary myoblasts (arrow) upon addition of Yoda1 and ionomycin. Representative traces ( c ), quantification of Yoda1-induced Ca 2+ influx ( d ) and basal Ca 2+ level ( e ) in b . **** P
Figure Legend Snippet: Impaired PIEZO1 activation in PS-exposing human primary myoblasts during myotube formation. a Detection of cell surface-exposed PS on differentiating human primary myoblasts by annexin V-GFP (PS, green), DAPI (nuclei, blue) and fixable viability dye (dead cells, red). b Fura2 imaging of Ca 2+ influx in annexin V-GFP-labelled human primary myoblasts (arrow) upon addition of Yoda1 and ionomycin. Representative traces ( c ), quantification of Yoda1-induced Ca 2+ influx ( d ) and basal Ca 2+ level ( e ) in b . **** P

Techniques Used: Activation Assay, Imaging

Impaired PIEZO1 activation in PS flippase-deficient myoblasts. a , b Suppression of agonist-induced PIEZO1 activation by PS flippase deficiency. a Left: Fura2 ratiometric measurements (F 340 /F 380 ) of Yoda1-induced Ca 2+ influx in WT, PIEZO1-deficient, CDC50A-deficient, and ATP11A-deficient C2C12 myoblasts. Right: quantification of Yoda1-induced Ca 2+ influx as the maximal increment of F 340 /F 380 (∆ ratio) in left. b Left: Fura2 ratiometric measurements of Yoda1-induced Ca 2+ influx in human primary myoblasts transfected with control, PIEZO1, CDC50A or ATP11A siRNA. Right: quantification of Yoda1-induced Ca 2+ influx in left. c Rescue of agonist-induced PIEZO1 activation in PS flippase-deficient myoblasts by overexpression of PS flippase complex components. Quantification of Yoda1-induced Ca 2+ influx in CDC50A-deficient (left) or ATP11A-deficient (right) C2C12 myoblasts expressing FLAG-tagged CDC50A, ATP11A, ATP11B or ATP11C. d , e Normal plasma membrane tension in PS flippase-deficient myoblasts. d Schematic model of membrane tension measurement using an optical trap. e Quantification of membrane tension in WT, CDC50A-deficient and cytochalasin D-treated WT C2C12 cells. f Schematic model showing PS flippase-mediated inward translocation of cell surface-exposed PS as a prerequisite for PIEZO1 activation. ** P
Figure Legend Snippet: Impaired PIEZO1 activation in PS flippase-deficient myoblasts. a , b Suppression of agonist-induced PIEZO1 activation by PS flippase deficiency. a Left: Fura2 ratiometric measurements (F 340 /F 380 ) of Yoda1-induced Ca 2+ influx in WT, PIEZO1-deficient, CDC50A-deficient, and ATP11A-deficient C2C12 myoblasts. Right: quantification of Yoda1-induced Ca 2+ influx as the maximal increment of F 340 /F 380 (∆ ratio) in left. b Left: Fura2 ratiometric measurements of Yoda1-induced Ca 2+ influx in human primary myoblasts transfected with control, PIEZO1, CDC50A or ATP11A siRNA. Right: quantification of Yoda1-induced Ca 2+ influx in left. c Rescue of agonist-induced PIEZO1 activation in PS flippase-deficient myoblasts by overexpression of PS flippase complex components. Quantification of Yoda1-induced Ca 2+ influx in CDC50A-deficient (left) or ATP11A-deficient (right) C2C12 myoblasts expressing FLAG-tagged CDC50A, ATP11A, ATP11B or ATP11C. d , e Normal plasma membrane tension in PS flippase-deficient myoblasts. d Schematic model of membrane tension measurement using an optical trap. e Quantification of membrane tension in WT, CDC50A-deficient and cytochalasin D-treated WT C2C12 cells. f Schematic model showing PS flippase-mediated inward translocation of cell surface-exposed PS as a prerequisite for PIEZO1 activation. ** P

Techniques Used: Activation Assay, Transfection, Over Expression, Expressing, Translocation Assay

Related Articles

Concentration Assay:

Article Title: Interactions between ?CaMKII and calmodulin in living cells: conformational changes arising from CaM -dependent and -independent relationships
Article Snippet: .. 5–10 cells were analyzed in each group. (C) Imaging of changes in Ca2+ concentration using Fura2-AM when HBSS perfusion was performed at 1 min following ionomycin application. ..

Incubation:

Article Title: Cell surface flip-flop of phosphatidylserine is critical for PIEZO1-mediated myotube formation
Article Snippet: .. For Ca2+ measurements, human primary myoblasts were differentiated on Cell Desk LF1 (Sumitomo Bakelite) for 2 days, incubated with Fura2-AM (15 μM, Dojindo) at RT for 30 min, washed with binding buffer and incubated with annexin V-EGFP at RT for 5 min. .. F2N12S measurements C2C12 cells were detached, suspended in HBSS and placed on ice for at least 10 min. Then, the cells (0.1−1 × 107 cells/mL) were incubated with F2N12S (0.1 μM, Thermo) for 5 min at 15 °C.

other:

Article Title: Evaluation of effects of various drugs on platelet functions using phorbol 12-myristate 13-acetate-induced megakaryocytic human erythroid leukemia cells
Article Snippet: Fura2-AM was purchased from Dojindo Laboratories (Kumamoto, Japan).

Article Title: Cell surface flip-flop of phosphatidylserine is critical for PIEZO1-mediated myotube formation
Article Snippet: Cells were loaded with Fura2-AM (5 μM) in 10% FBS/DMEM at 37 °C for 40 min and washed with HEPES-buffered saline (HBS) .

Binding Assay:

Article Title: Cell surface flip-flop of phosphatidylserine is critical for PIEZO1-mediated myotube formation
Article Snippet: .. For Ca2+ measurements, human primary myoblasts were differentiated on Cell Desk LF1 (Sumitomo Bakelite) for 2 days, incubated with Fura2-AM (15 μM, Dojindo) at RT for 30 min, washed with binding buffer and incubated with annexin V-EGFP at RT for 5 min. .. F2N12S measurements C2C12 cells were detached, suspended in HBSS and placed on ice for at least 10 min. Then, the cells (0.1−1 × 107 cells/mL) were incubated with F2N12S (0.1 μM, Thermo) for 5 min at 15 °C.

Imaging:

Article Title: Interactions between ?CaMKII and calmodulin in living cells: conformational changes arising from CaM -dependent and -independent relationships
Article Snippet: .. Ca2+ imaging Cells were loaded with Fura2-AM (1 μg, DOJINDO) at 37°C for 30 min, and then washed four times with HBSS. .. Fluorescence was monitored as above (FRET analysis) using specific filters (CHROMA).

Article Title: Interactions between ?CaMKII and calmodulin in living cells: conformational changes arising from CaM -dependent and -independent relationships
Article Snippet: .. 5–10 cells were analyzed in each group. (C) Imaging of changes in Ca2+ concentration using Fura2-AM when HBSS perfusion was performed at 1 min following ionomycin application. ..

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 92
    Dojindo Labs fura2 am
    Effect of ibuprofen or sodium valproate on [Ca 2+ ] i induced by thrombin. Notes: <t>Fura2-loaded</t> untreated HEL cells and PMA-induced HEL cells were treated with ibuprofen ( A ; 0.8–200 µM) or sodium valproate ( B ; 50–1,000 µg/mL) for 6 minutes, then stimulated with thrombin. ■, untreated HEL cells; □, PMA-induced HEL cells. Intracellular Ca 2+ mobilization was induced by thrombin. Results are presented as the mean ± SD, n=5. ** P
    Fura2 Am, supplied by Dojindo Labs, used in various techniques. Bioz Stars score: 92/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fura2 am/product/Dojindo Labs
    Average 92 stars, based on 9 article reviews
    Price from $9.99 to $1999.99
    fura2 am - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    Image Search Results


    Effect of ibuprofen or sodium valproate on [Ca 2+ ] i induced by thrombin. Notes: Fura2-loaded untreated HEL cells and PMA-induced HEL cells were treated with ibuprofen ( A ; 0.8–200 µM) or sodium valproate ( B ; 50–1,000 µg/mL) for 6 minutes, then stimulated with thrombin. ■, untreated HEL cells; □, PMA-induced HEL cells. Intracellular Ca 2+ mobilization was induced by thrombin. Results are presented as the mean ± SD, n=5. ** P

    Journal: Drug Design, Development and Therapy

    Article Title: Evaluation of effects of various drugs on platelet functions using phorbol 12-myristate 13-acetate-induced megakaryocytic human erythroid leukemia cells

    doi: 10.2147/DDDT.S115910

    Figure Lengend Snippet: Effect of ibuprofen or sodium valproate on [Ca 2+ ] i induced by thrombin. Notes: Fura2-loaded untreated HEL cells and PMA-induced HEL cells were treated with ibuprofen ( A ; 0.8–200 µM) or sodium valproate ( B ; 50–1,000 µg/mL) for 6 minutes, then stimulated with thrombin. ■, untreated HEL cells; □, PMA-induced HEL cells. Intracellular Ca 2+ mobilization was induced by thrombin. Results are presented as the mean ± SD, n=5. ** P

    Article Snippet: Fura2-AM was purchased from Dojindo Laboratories (Kumamoto, Japan).

    Techniques:

    Effect of aspirin or cilostazol on [Ca 2+ ] i induced by thrombin. Notes: Fura2-loaded untreated HEL cells and PMA-induced HEL cells were treated with aspirin ( A ; 5.6–560 µM) or cilostazol ( B ; 1–10 µM) for 6 minutes and then stimulated with thrombin. ■, untreated HEL cells; □, PMA-induced HEL cells. Intracellular Ca 2+ mobilization was induced by thrombin. Results are presented as the mean ± SD, n=5. ** P

    Journal: Drug Design, Development and Therapy

    Article Title: Evaluation of effects of various drugs on platelet functions using phorbol 12-myristate 13-acetate-induced megakaryocytic human erythroid leukemia cells

    doi: 10.2147/DDDT.S115910

    Figure Lengend Snippet: Effect of aspirin or cilostazol on [Ca 2+ ] i induced by thrombin. Notes: Fura2-loaded untreated HEL cells and PMA-induced HEL cells were treated with aspirin ( A ; 5.6–560 µM) or cilostazol ( B ; 1–10 µM) for 6 minutes and then stimulated with thrombin. ■, untreated HEL cells; □, PMA-induced HEL cells. Intracellular Ca 2+ mobilization was induced by thrombin. Results are presented as the mean ± SD, n=5. ** P

    Article Snippet: Fura2-AM was purchased from Dojindo Laboratories (Kumamoto, Japan).

    Techniques:

    Effect of drug combinations on [Ca 2+ ] i induced by thrombin. Notes: Fura2-loaded PMA-induced HEL cells were treated with the simultaneous combined use of aspirin (ASA) and ibuprofen (IBU) ( A and B ) or sodium valproate (VPA) ( C ) for 6 minutes, then stimulated with thrombin. Intracellular Ca 2+ mobilization was induced by thrombin. Results are presented as the mean ± SD, n=5. ** P

    Journal: Drug Design, Development and Therapy

    Article Title: Evaluation of effects of various drugs on platelet functions using phorbol 12-myristate 13-acetate-induced megakaryocytic human erythroid leukemia cells

    doi: 10.2147/DDDT.S115910

    Figure Lengend Snippet: Effect of drug combinations on [Ca 2+ ] i induced by thrombin. Notes: Fura2-loaded PMA-induced HEL cells were treated with the simultaneous combined use of aspirin (ASA) and ibuprofen (IBU) ( A and B ) or sodium valproate (VPA) ( C ) for 6 minutes, then stimulated with thrombin. Intracellular Ca 2+ mobilization was induced by thrombin. Results are presented as the mean ± SD, n=5. ** P

    Article Snippet: Fura2-AM was purchased from Dojindo Laboratories (Kumamoto, Japan).

    Techniques:

    Impaired PIEZO1 activation in PS-exposing human primary myoblasts during myotube formation. a Detection of cell surface-exposed PS on differentiating human primary myoblasts by annexin V-GFP (PS, green), DAPI (nuclei, blue) and fixable viability dye (dead cells, red). b Fura2 imaging of Ca 2+ influx in annexin V-GFP-labelled human primary myoblasts (arrow) upon addition of Yoda1 and ionomycin. Representative traces ( c ), quantification of Yoda1-induced Ca 2+ influx ( d ) and basal Ca 2+ level ( e ) in b . **** P

    Journal: Nature Communications

    Article Title: Cell surface flip-flop of phosphatidylserine is critical for PIEZO1-mediated myotube formation

    doi: 10.1038/s41467-018-04436-w

    Figure Lengend Snippet: Impaired PIEZO1 activation in PS-exposing human primary myoblasts during myotube formation. a Detection of cell surface-exposed PS on differentiating human primary myoblasts by annexin V-GFP (PS, green), DAPI (nuclei, blue) and fixable viability dye (dead cells, red). b Fura2 imaging of Ca 2+ influx in annexin V-GFP-labelled human primary myoblasts (arrow) upon addition of Yoda1 and ionomycin. Representative traces ( c ), quantification of Yoda1-induced Ca 2+ influx ( d ) and basal Ca 2+ level ( e ) in b . **** P

    Article Snippet: Cells were loaded with Fura2-AM (5 μM) in 10% FBS/DMEM at 37 °C for 40 min and washed with HEPES-buffered saline (HBS) .

    Techniques: Activation Assay, Imaging

    Impaired PIEZO1 activation in PS flippase-deficient myoblasts. a , b Suppression of agonist-induced PIEZO1 activation by PS flippase deficiency. a Left: Fura2 ratiometric measurements (F 340 /F 380 ) of Yoda1-induced Ca 2+ influx in WT, PIEZO1-deficient, CDC50A-deficient, and ATP11A-deficient C2C12 myoblasts. Right: quantification of Yoda1-induced Ca 2+ influx as the maximal increment of F 340 /F 380 (∆ ratio) in left. b Left: Fura2 ratiometric measurements of Yoda1-induced Ca 2+ influx in human primary myoblasts transfected with control, PIEZO1, CDC50A or ATP11A siRNA. Right: quantification of Yoda1-induced Ca 2+ influx in left. c Rescue of agonist-induced PIEZO1 activation in PS flippase-deficient myoblasts by overexpression of PS flippase complex components. Quantification of Yoda1-induced Ca 2+ influx in CDC50A-deficient (left) or ATP11A-deficient (right) C2C12 myoblasts expressing FLAG-tagged CDC50A, ATP11A, ATP11B or ATP11C. d , e Normal plasma membrane tension in PS flippase-deficient myoblasts. d Schematic model of membrane tension measurement using an optical trap. e Quantification of membrane tension in WT, CDC50A-deficient and cytochalasin D-treated WT C2C12 cells. f Schematic model showing PS flippase-mediated inward translocation of cell surface-exposed PS as a prerequisite for PIEZO1 activation. ** P

    Journal: Nature Communications

    Article Title: Cell surface flip-flop of phosphatidylserine is critical for PIEZO1-mediated myotube formation

    doi: 10.1038/s41467-018-04436-w

    Figure Lengend Snippet: Impaired PIEZO1 activation in PS flippase-deficient myoblasts. a , b Suppression of agonist-induced PIEZO1 activation by PS flippase deficiency. a Left: Fura2 ratiometric measurements (F 340 /F 380 ) of Yoda1-induced Ca 2+ influx in WT, PIEZO1-deficient, CDC50A-deficient, and ATP11A-deficient C2C12 myoblasts. Right: quantification of Yoda1-induced Ca 2+ influx as the maximal increment of F 340 /F 380 (∆ ratio) in left. b Left: Fura2 ratiometric measurements of Yoda1-induced Ca 2+ influx in human primary myoblasts transfected with control, PIEZO1, CDC50A or ATP11A siRNA. Right: quantification of Yoda1-induced Ca 2+ influx in left. c Rescue of agonist-induced PIEZO1 activation in PS flippase-deficient myoblasts by overexpression of PS flippase complex components. Quantification of Yoda1-induced Ca 2+ influx in CDC50A-deficient (left) or ATP11A-deficient (right) C2C12 myoblasts expressing FLAG-tagged CDC50A, ATP11A, ATP11B or ATP11C. d , e Normal plasma membrane tension in PS flippase-deficient myoblasts. d Schematic model of membrane tension measurement using an optical trap. e Quantification of membrane tension in WT, CDC50A-deficient and cytochalasin D-treated WT C2C12 cells. f Schematic model showing PS flippase-mediated inward translocation of cell surface-exposed PS as a prerequisite for PIEZO1 activation. ** P

    Article Snippet: Cells were loaded with Fura2-AM (5 μM) in 10% FBS/DMEM at 37 °C for 40 min and washed with HEPES-buffered saline (HBS) .

    Techniques: Activation Assay, Transfection, Over Expression, Expressing, Translocation Assay

    Time lapse imaging of αCaMKII activation in HeLa cells. (A) Changes in intracellular Ca 2+ concentration measured using Fura2-AM. Upper panels show images of typical 340 nm/380 nm ratio at each time point before and after ionomycin application. Lower graph shows changes in relative 340 nm/380 nm ratio (open circle, ionomycin; filled circle, DMSO). Black bar shows the period of ionomycin application. 12–18 cells were analyzed in each group. (B, C) Changes in the interaction of YFP-αCaMKII with CFP-CaM (B) and the conformational change of YFP-αCaMKII-CFP (C) . Upper panels show images of typical YFP/CFP emission ratio before and after ionomycin application (upper images, –KN-93; lower images, +KN-93). Lower graphs show changes in relative YFP/CFP emission ratio (open circle, ionomycin; filled circle, ionomycin with KN-93). Black bar shows the period of ionomycin application. 9–13 cells were analyzed in each group. (D) Effect of removal of ionomycin by wash out at 0.5 or 1 min following ionomycin application. Upper graph shows the interaction of YFP-αCaMKII with CFP-CaM (open circle, –wash; triangle, 0.5 min; filled circle, 1 min). Lower graph shows the conformational change of YFP-αCaMKII-CFP (open circle, -wash; triangle, 0.5 min; filled circle, 1 min). 10–12 cells were analyzed in each group. (E) Auto-phosphorylation at T286 of αCaMKII fusion proteins after ionomycin application at each time point (0, 1, 5 and 10 min). Upper panel shows Western blot analyses analyzing the expression level of phospho-αCaMKII fusion proteins and total-αCaMKII fusion proteins. Lower graph shows relative ratio of phospho-αCaMKII/αCaMKII levels (n = 3).

    Journal: Molecular Brain

    Article Title: Interactions between ?CaMKII and calmodulin in living cells: conformational changes arising from CaM -dependent and -independent relationships

    doi: 10.1186/1756-6606-6-37

    Figure Lengend Snippet: Time lapse imaging of αCaMKII activation in HeLa cells. (A) Changes in intracellular Ca 2+ concentration measured using Fura2-AM. Upper panels show images of typical 340 nm/380 nm ratio at each time point before and after ionomycin application. Lower graph shows changes in relative 340 nm/380 nm ratio (open circle, ionomycin; filled circle, DMSO). Black bar shows the period of ionomycin application. 12–18 cells were analyzed in each group. (B, C) Changes in the interaction of YFP-αCaMKII with CFP-CaM (B) and the conformational change of YFP-αCaMKII-CFP (C) . Upper panels show images of typical YFP/CFP emission ratio before and after ionomycin application (upper images, –KN-93; lower images, +KN-93). Lower graphs show changes in relative YFP/CFP emission ratio (open circle, ionomycin; filled circle, ionomycin with KN-93). Black bar shows the period of ionomycin application. 9–13 cells were analyzed in each group. (D) Effect of removal of ionomycin by wash out at 0.5 or 1 min following ionomycin application. Upper graph shows the interaction of YFP-αCaMKII with CFP-CaM (open circle, –wash; triangle, 0.5 min; filled circle, 1 min). Lower graph shows the conformational change of YFP-αCaMKII-CFP (open circle, -wash; triangle, 0.5 min; filled circle, 1 min). 10–12 cells were analyzed in each group. (E) Auto-phosphorylation at T286 of αCaMKII fusion proteins after ionomycin application at each time point (0, 1, 5 and 10 min). Upper panel shows Western blot analyses analyzing the expression level of phospho-αCaMKII fusion proteins and total-αCaMKII fusion proteins. Lower graph shows relative ratio of phospho-αCaMKII/αCaMKII levels (n = 3).

    Article Snippet: 5–10 cells were analyzed in each group. (C) Imaging of changes in Ca2+ concentration using Fura2-AM when HBSS perfusion was performed at 1 min following ionomycin application.

    Techniques: Imaging, Activation Assay, Concentration Assay, Chick Chorioallantoic Membrane Assay, Western Blot, Expressing

    Time lapse imaging of αCaMKII activation in SH-SY5Y cells. (A) Changes in intracellular Ca 2+ concentration measured using Fura2-AM. Upper panels show images of typical 340 nm/380 nm ratio at each time point before and after the application of ionomycin or KCl (50 mM)-application. Lower graph shows changes in relative 340 nm/380 nm ratio (open circle, ionomycin; filled circle, KCl). Black bar shows the period of ionomycin or KCl application. 30–40 cells were analyzed in each group. (B, C) Changes in the interaction of YFP-αCaMKII with CFP-CaM (B) and the conformational change of YFP-αCaMKII-CFP (C) . Upper panels show images of typical YFP/CFP emission ratio before and after ionomycin or KCl application. Lower graphs show changes in relative YFP/CFP emission ratio (open circle, ionomycin; filled circle, KCl). Black bar shows the period of ionomycin or KCl application. 11–25 cells were analyzed in each group. (D) Auto-phosphorylation at T286 of αCaMKII fusion proteins after ionomycin or KCI application at each time point (0, 1, 5 and 10 min). Upper panel shows Western blot analyses analyzing the expression level of phospho-αCaMKII fusion proteins and total-αCaMKII fusion proteins. Lower graph shows relative ratio of phospho-αCaMKII/αCaMKII levels (n = 3).

    Journal: Molecular Brain

    Article Title: Interactions between ?CaMKII and calmodulin in living cells: conformational changes arising from CaM -dependent and -independent relationships

    doi: 10.1186/1756-6606-6-37

    Figure Lengend Snippet: Time lapse imaging of αCaMKII activation in SH-SY5Y cells. (A) Changes in intracellular Ca 2+ concentration measured using Fura2-AM. Upper panels show images of typical 340 nm/380 nm ratio at each time point before and after the application of ionomycin or KCl (50 mM)-application. Lower graph shows changes in relative 340 nm/380 nm ratio (open circle, ionomycin; filled circle, KCl). Black bar shows the period of ionomycin or KCl application. 30–40 cells were analyzed in each group. (B, C) Changes in the interaction of YFP-αCaMKII with CFP-CaM (B) and the conformational change of YFP-αCaMKII-CFP (C) . Upper panels show images of typical YFP/CFP emission ratio before and after ionomycin or KCl application. Lower graphs show changes in relative YFP/CFP emission ratio (open circle, ionomycin; filled circle, KCl). Black bar shows the period of ionomycin or KCl application. 11–25 cells were analyzed in each group. (D) Auto-phosphorylation at T286 of αCaMKII fusion proteins after ionomycin or KCI application at each time point (0, 1, 5 and 10 min). Upper panel shows Western blot analyses analyzing the expression level of phospho-αCaMKII fusion proteins and total-αCaMKII fusion proteins. Lower graph shows relative ratio of phospho-αCaMKII/αCaMKII levels (n = 3).

    Article Snippet: 5–10 cells were analyzed in each group. (C) Imaging of changes in Ca2+ concentration using Fura2-AM when HBSS perfusion was performed at 1 min following ionomycin application.

    Techniques: Imaging, Activation Assay, Concentration Assay, Chick Chorioallantoic Membrane Assay, Western Blot, Expressing

    Impaired PIEZO1 activation in PS-exposing human primary myoblasts during myotube formation. a Detection of cell surface-exposed PS on differentiating human primary myoblasts by annexin V-GFP (PS, green), DAPI (nuclei, blue) and fixable viability dye (dead cells, red). b Fura2 imaging of Ca 2+ influx in annexin V-GFP-labelled human primary myoblasts (arrow) upon addition of Yoda1 and ionomycin. Representative traces ( c ), quantification of Yoda1-induced Ca 2+ influx ( d ) and basal Ca 2+ level ( e ) in b . **** P

    Journal: Nature Communications

    Article Title: Cell surface flip-flop of phosphatidylserine is critical for PIEZO1-mediated myotube formation

    doi: 10.1038/s41467-018-04436-w

    Figure Lengend Snippet: Impaired PIEZO1 activation in PS-exposing human primary myoblasts during myotube formation. a Detection of cell surface-exposed PS on differentiating human primary myoblasts by annexin V-GFP (PS, green), DAPI (nuclei, blue) and fixable viability dye (dead cells, red). b Fura2 imaging of Ca 2+ influx in annexin V-GFP-labelled human primary myoblasts (arrow) upon addition of Yoda1 and ionomycin. Representative traces ( c ), quantification of Yoda1-induced Ca 2+ influx ( d ) and basal Ca 2+ level ( e ) in b . **** P

    Article Snippet: Cells were loaded with Fura2-AM (5 μM) in 10% FBS/DMEM at 37 °C for 40 min and washed with HEPES-buffered saline (HBS) .

    Techniques: Activation Assay, Imaging

    Impaired PIEZO1 activation in PS flippase-deficient myoblasts. a , b Suppression of agonist-induced PIEZO1 activation by PS flippase deficiency. a Left: Fura2 ratiometric measurements (F 340 /F 380 ) of Yoda1-induced Ca 2+ influx in WT, PIEZO1-deficient, CDC50A-deficient, and ATP11A-deficient C2C12 myoblasts. Right: quantification of Yoda1-induced Ca 2+ influx as the maximal increment of F 340 /F 380 (∆ ratio) in left. b Left: Fura2 ratiometric measurements of Yoda1-induced Ca 2+ influx in human primary myoblasts transfected with control, PIEZO1, CDC50A or ATP11A siRNA. Right: quantification of Yoda1-induced Ca 2+ influx in left. c Rescue of agonist-induced PIEZO1 activation in PS flippase-deficient myoblasts by overexpression of PS flippase complex components. Quantification of Yoda1-induced Ca 2+ influx in CDC50A-deficient (left) or ATP11A-deficient (right) C2C12 myoblasts expressing FLAG-tagged CDC50A, ATP11A, ATP11B or ATP11C. d , e Normal plasma membrane tension in PS flippase-deficient myoblasts. d Schematic model of membrane tension measurement using an optical trap. e Quantification of membrane tension in WT, CDC50A-deficient and cytochalasin D-treated WT C2C12 cells. f Schematic model showing PS flippase-mediated inward translocation of cell surface-exposed PS as a prerequisite for PIEZO1 activation. ** P

    Journal: Nature Communications

    Article Title: Cell surface flip-flop of phosphatidylserine is critical for PIEZO1-mediated myotube formation

    doi: 10.1038/s41467-018-04436-w

    Figure Lengend Snippet: Impaired PIEZO1 activation in PS flippase-deficient myoblasts. a , b Suppression of agonist-induced PIEZO1 activation by PS flippase deficiency. a Left: Fura2 ratiometric measurements (F 340 /F 380 ) of Yoda1-induced Ca 2+ influx in WT, PIEZO1-deficient, CDC50A-deficient, and ATP11A-deficient C2C12 myoblasts. Right: quantification of Yoda1-induced Ca 2+ influx as the maximal increment of F 340 /F 380 (∆ ratio) in left. b Left: Fura2 ratiometric measurements of Yoda1-induced Ca 2+ influx in human primary myoblasts transfected with control, PIEZO1, CDC50A or ATP11A siRNA. Right: quantification of Yoda1-induced Ca 2+ influx in left. c Rescue of agonist-induced PIEZO1 activation in PS flippase-deficient myoblasts by overexpression of PS flippase complex components. Quantification of Yoda1-induced Ca 2+ influx in CDC50A-deficient (left) or ATP11A-deficient (right) C2C12 myoblasts expressing FLAG-tagged CDC50A, ATP11A, ATP11B or ATP11C. d , e Normal plasma membrane tension in PS flippase-deficient myoblasts. d Schematic model of membrane tension measurement using an optical trap. e Quantification of membrane tension in WT, CDC50A-deficient and cytochalasin D-treated WT C2C12 cells. f Schematic model showing PS flippase-mediated inward translocation of cell surface-exposed PS as a prerequisite for PIEZO1 activation. ** P

    Article Snippet: Cells were loaded with Fura2-AM (5 μM) in 10% FBS/DMEM at 37 °C for 40 min and washed with HEPES-buffered saline (HBS) .

    Techniques: Activation Assay, Transfection, Over Expression, Expressing, Translocation Assay