ca 2 sensitive fluorescence indicator fluo  (Biotium)

Journal: Aging Cell

Article Title: Piezo1 exacerbates inflammation‐induced cartilaginous endplate degeneration by activating mitochondrial fission via the Ca 2+ / CaMKII /Drp1 axis

doi: 10.1111/acel.14440

Figure Lengend Snippet: Piezo1 promoted cartilaginous endplate (CEP) cell senescence and apoptosis by triggering mitochondrial fragmentation and dysfunction. (a, f) The influences of Yoda1 and GsMTX4 on intracellular Ca 2+ levels in LPS‐treated CEP cells were using the specific Ca 2+ ‐sensitive fluorescent indicator Fluo‐4 AM. Scale bar, 100 μm. (b) The expression of apoptosis‐related proteins (cleaved caspase‐3, Bax, Bcl‐2) was measured by western blotting. (c) The expression of senescence‐related proteins (p53, p21 and p16) was measured by western blotting. (d, g) The effects of Yoda1 and GsMTX4 on LPS‐induced apoptosis of CEP cells were analysed by flow cytometry with Annexin V‐FITC/PI. (e, h) Flow cytometry with β‐galactosidase staining was used to evaluate the level of CEP cell senescence. (i) The mitochondrial morphology in CEP cells was detected by MitoTracker Red staining. Scale bar, 25 μm. (j, m) The MMP in CEP cells was assessed by flow cytometry using JC‐1 staining. (k, l, n and o) The production of mitochondrial and cellular reactive oxygen species (ROS) in CEP cells was measured by MitoSOX Red and DCFH‐DA staining, respectively. Scale bars, 50 μm (k) and 100 μm (l). (p) Relative ATP contents in LPS‐induced CEP cells after treated with Yoda1 or GsMTx4. ( n = 3 biological replicates, * p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: Intracellular Ca 2+ levels were detected using the specific Ca 2+ ‐sensitive fluorescent indicator Fluo‐4 AM (F14201, Thermo Fisher Scientific) according to the manufacturer's instructions.

Techniques: Expressing, Western Blot, Flow Cytometry, Staining

Journal: Aging Cell

Article Title: Piezo1 exacerbates inflammation‐induced cartilaginous endplate degeneration by activating mitochondrial fission via the Ca 2+ / CaMKII /Drp1 axis

doi: 10.1111/acel.14440

Figure Lengend Snippet: Piezo1 induced mitochondrial fission and dysfunction via the Ca 2+ /CaMKII/Drp1 axis in LPS‐treated cartilaginous endplate (CEP) cells. (a–e) The effects of KN‐93 on the phosphorylation and mitochondrial translocation of Drp1 were assessed using western blotting. (f, g, l, m) TUNEL staining and flow cytometry with Annexin V‐FITC/PI demonstrated that Mdivi‐1 partially rescued Yoda1‐induced CEP cell apoptosis. Scale bar, 100 μm. (h) The colocalization between p‐Drp1 and MitoTracker Red was verified by double immunofluorescence staining. Scale bar, 25 μm. (i, n) The MMP in CEP cells was evaluated by flow cytometry using JC‐1. (j, k, o, p) The production of mitochondrial and cellular reactive oxygen species (ROS) in CEP cells was measured by MitoSOX Red and DCFH‐DA staining, respectively. Scale bars, 50 μm (j) and 100 μm (k). ( n = 3 biological replicates, * p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: Intracellular Ca 2+ levels were detected using the specific Ca 2+ ‐sensitive fluorescent indicator Fluo‐4 AM (F14201, Thermo Fisher Scientific) according to the manufacturer's instructions.

Techniques: Phospho-proteomics, Translocation Assay, Western Blot, TUNEL Assay, Staining, Flow Cytometry, Double Immunofluorescence Staining

Journal: Aging Cell

Article Title: Piezo1 exacerbates inflammation‐induced cartilaginous endplate degeneration by activating mitochondrial fission via the Ca 2+ / CaMKII /Drp1 axis

doi: 10.1111/acel.14440

Figure Lengend Snippet: Knockdown of Piezo1 inhibited Drp1‐dependent mitochondrial fission. (a) The level of CaMKII phosphorylation and Drp1 phosphorylation after si‐Piezo1 treatment was measured by western blot analysis. (b) The subcellular localization of Drp1 was detected by western blotting. (c, d) The expression levels of apoptosis‐related and senescence‐related proteins were measured by western blot analysis. (e) The colocalization of Drp1 and mitochondria was shown by immunofluorescence staining and confocal microscopy. Scale bar, 10 μm. (f) The mitochondrial morphology was demonstrated by MitoTracker Red staining. Scale bar, 10 μm. (g, h) The intracellular Ca 2+ levels were analysed by using the specific Ca 2+ ‐sensitive fluorescent indicator Fluo‐4 AM. Scale bar, 40 μm. (i–l) The accumulation of mitochondrial and cellular reactive oxygen species (ROS) in cartilaginous endplate (CEP) cells was measured by MitoSOX Red and DCFH‐DA staining, respectively. Scale bars, 40 μm (i) and 40 μm (k). (m) Relative adenosine triphosphate (ATP) productions in CEP cells treated by si‐Piezo1. (n) The MMP was evaluated by flow cytometry using JC‐1. (o) Flow cytometry with Annexin V‐FITC/PI showed that si‐Piezo1 partially rescued LPS‐induced CEP cell apoptosis. (p) Cellular senescence was analysed by using flow cytometry with β‐galactosidase staining. ( n = 3 biological replicates, * p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: Intracellular Ca 2+ levels were detected using the specific Ca 2+ ‐sensitive fluorescent indicator Fluo‐4 AM (F14201, Thermo Fisher Scientific) according to the manufacturer's instructions.

Techniques: Knockdown, Phospho-proteomics, Western Blot, Expressing, Immunofluorescence, Staining, Confocal Microscopy, Flow Cytometry

Journal: FEBS Open Bio

Article Title: MFN2 deficiency affects calcium homeostasis in lung adenocarcinoma cells via downregulation of UCP4

doi: 10.1002/2211-5463.13591

Figure Lengend Snippet: UCP4 overexpression regulates the intracellular calcium concentration in MFN2‐KO A549 cells. (A) qRT–PCR and western blot analysis of UCP4 and MFN2 expression in MFN2‐KO A549 cells stably transfected with UCP4‐HA or MFN2‐FLAG. (B) The mtDNA copy number in MFN2‐KO A549 cells transduced with UCP4‐HA or MFN2‐FLAG was determined by qRT–PCR. (C) The ROS levels in MFN2‐KO A549 cells transduced with UCP4‐HA or MFN2‐FLAG were measured using H 2 DCFDA by flow cytometry. Representative histograms are shown on the left, and a bar graph of the mean fluorescence intensity is displayed on the right. (D, E) The MMP of MFN2‐KO A549 cells transduced with UCP4‐HA or MFN2‐FLAG was determined by JC‐1 staining. (D) shows the proportion of cells with low MMP. (E) indicating representative flow cytometry pictures. (F) The ATP contents in MFN2‐KO A549 cells were measured by an ATP Assay Kit. (G) The Ca 2+ levels in MFN2‐KO A549 cells overexpressing UCP4‐HA or MFN2‐FLAG were quantified by flow cytometry (histograms on the left). The mean Fluo‐4 AM fluorescence intensity is shown on the right. (H) qRT–PCR analysis of genes related to the mitochondrial respiratory chain complex in MFN2‐KO A549 cells. * P < 0.05; ** P < 0.01; *** P < 0.001; n.s., nonsignificant (Student's t ‐test). Values were mean ± SEM of three independent experiments.

Article Snippet: To detect the intracellular calcium levels, the cells were stained for 30–40 min at 37 °C with the Ca 2+ ‐sensitive fluorescent indicator Fluo‐4 AM (2 μ m , S1060, Beyotime, Shanghai, China) or Fura‐2 AM (5 μ m , S1052, Beyotime).

Techniques: Over Expression, Concentration Assay, Quantitative RT-PCR, Western Blot, Expressing, Stable Transfection, Transfection, Transduction, Flow Cytometry, Fluorescence, Staining, ATP Assay

Journal: FEBS Open Bio

Article Title: MFN2 deficiency affects calcium homeostasis in lung adenocarcinoma cells via downregulation of UCP4

doi: 10.1002/2211-5463.13591

Figure Lengend Snippet: UCP4 overexpression regulates the intracellular calcium concentration in MFN2‐KD H1975 cells. (A) qRT–PCR and western blot analysis of MFN2 expression in 293T cells transduced with MFN2 shRNA#1‐#3. (B) Western blot analysis of MFN2 and UCP4 expression in scramble and MFN2‐KD H1975 cells. (C) qRT–PCR and western blot analysis of UCP4 and MFN2 expression in MFN2‐KD H1975 cells stably transfected with UCP4‐HA or MFN2‐FLAG. (D) qRT–PCR analysis of genes related to the mitochondrial respiratory chain complex in MFN2‐KD H1975 cells. (E) The ATP contents in MFN2‐KD H1975 cells overexpressing UCP4‐HA or MFN2‐FLAG were measured by ATP Assay Kit. (F) The Ca 2+ levels in MFN2‐KD H1975 cells overexpressing UCP4‐HA or MFN2‐FLAG were quantified by flow cytometry (histograms on the left). The mean Fluo‐4 AM fluorescence intensity is shown on the right. * P < 0.05; ** P < 0.01; *** P < 0.001; n.s., nonsignificant (Student's t ‐test). Values were mean ± SEM of three independent experiments.

Article Snippet: To detect the intracellular calcium levels, the cells were stained for 30–40 min at 37 °C with the Ca 2+ ‐sensitive fluorescent indicator Fluo‐4 AM (2 μ m , S1060, Beyotime, Shanghai, China) or Fura‐2 AM (5 μ m , S1052, Beyotime).

Techniques: Over Expression, Concentration Assay, Quantitative RT-PCR, Western Blot, Expressing, Transduction, shRNA, Stable Transfection, Transfection, ATP Assay, Flow Cytometry, Fluorescence

Journal: FEBS Open Bio

Article Title: MFN2 deficiency affects calcium homeostasis in lung adenocarcinoma cells via downregulation of UCP4

doi: 10.1002/2211-5463.13591

Figure Lengend Snippet: Protein–protein interaction network of MFN2/UCP4 in the regulation of calcium channels. (A) Ca 2+ ‐related proteins KEGG pathway enrichment analyses. (B) PPI network of the proteins in A based on STRING. (C) Flowchart for screening calcium channels. (D) Venn diagram showing the overlap between the 460 overlapping proteins (pink) and calcium channels in PubMed (blue). (E) Heatmap of the eight overlapping genes. (F) PPI network of the proteins in (E) based on STRING.

Article Snippet: To detect the intracellular calcium levels, the cells were stained for 30–40 min at 37 °C with the Ca 2+ ‐sensitive fluorescent indicator Fluo‐4 AM (2 μ m , S1060, Beyotime, Shanghai, China) or Fura‐2 AM (5 μ m , S1052, Beyotime).

Techniques:

Journal: FEBS Open Bio

Article Title: MFN2 deficiency affects calcium homeostasis in lung adenocarcinoma cells via downregulation of UCP4

doi: 10.1002/2211-5463.13591

Figure Lengend Snippet: PINK1 increases the MFN2/UCP4‐mediated intracellular calcium concentration. (A, B) qRT–PCR and western blot analysis of PINK1 expression in MFN2‐KO A549 (A) and MFN2‐KD H1975 (B) cells. (C) qRT–PCR and western blot analysis of UCP4, MFN2, and PINK1 expression in 293T cells transduced with PINK1‐Myc. (D, E) Western blot analysis of UCP4, MFN2, and PINK1 expression in MFN2‐KO A549 (D) and MFN2‐KD H1975 (E) cells stably transfected with UCP4‐HA or PINK1‐Myc. (F, G) qRT–PCR analysis of UCP4, MFN2, and PINK1 expression in MFN2‐KO A549 (F) and MFN2‐KD H1975 (G) cells stably transfected with UCP4‐HA or PINK1‐Myc. (H, I) The Ca 2+ levels in MFN2‐KO A549 (H) and MFN2‐KD H1975 (I) cells were quantified by flow cytometry (histograms on the left). The mean Fluo‐4 AM fluorescence intensity is shown on the right. * P < 0.05; ** P < 0.01; *** P < 0.001; n.s., nonsignificant (Student's t ‐test). Values were mean ± SEM of three independent experiments.

Article Snippet: To detect the intracellular calcium levels, the cells were stained for 30–40 min at 37 °C with the Ca 2+ ‐sensitive fluorescent indicator Fluo‐4 AM (2 μ m , S1060, Beyotime, Shanghai, China) or Fura‐2 AM (5 μ m , S1052, Beyotime).

Techniques: Concentration Assay, Quantitative RT-PCR, Western Blot, Expressing, Transduction, Stable Transfection, Transfection, Flow Cytometry, Fluorescence

Journal: Archives of Toxicology

Article Title: Neurotoxicity of Tityus bahiensis (brown scorpion) venom in sympathetic vas deferens preparations and neuronal cells

doi: 10.1007/s00204-020-02799-y

Figure Lengend Snippet: Calcium mobilization in ND7-23 wt neurons incubated with T. bahiensis venom (0.3 µg/ml). Calcium movement was monitored in cells loaded with the Ca 2+ -sensitive fluorescent dye Fluo-4AM. Note the time-dependent increase in calcium mobilization from 50 min onwards. The lines in ( a ) and the cells in ( b ) are representative recordings from 11–22 cells in 5–7 experiments. Fluorescence scale (from low to high calcium concentration): light blue < dark blue < green < yellow < red. Magnification in b : 100 ×

Article Snippet: Intracellular Ca 2+ transients were monitored in ND7-23 wild type neurons using the Ca 2+ sensitive fluorescent indicator Fluo-4 AM (Biotium Inc., Hayward, CA, USA) prepared as a 1 mM stock solution in dimethyl sulfoxide (DMSO; Sigma-Aldrich) and stored at − 20 °C.

Techniques: Incubation, Fluorescence, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: β 2 -Adrenergic Receptors Chaperone Trapped Bitter Taste Receptor 14 to the Cell Surface as a Heterodimer and Exert Unidirectional Desensitization of Taste Receptor Function *

doi: 10.1074/jbc.M116.722736

Figure Lengend Snippet: TAS2R14 expressed on HEK-293T cells couple to [Ca 2+ ] i release. Cells transfected with Gα16/G44 with TAS2R14 and β 2 AR were loaded with Fluo-4 and [Ca 2+ ] i quantitated by fluorescent microscopy ( A and B ) or by a fluorescence based plate based assay (see “Experimental Procedures”) ( C ). Control pcDNA-transfected cells showed a minimal to no response to TAS2R agonists ( A and B ). TAS2R14 + β 2 AR cells responded to the TAS2R14 agonists quinine ( QUI ), DPD, and FFA, but not the TAS2R10 agonist strychnine or the TAS2R31 agonist saccharin. *, p < 0.01 versus pcDNA-transfected, n >500 cells imaged per condition. A representative DPD dose-response for stimulation of [Ca 2+ ] i is shown in C . Magnification = ×20, bar = 400 μm.

Article Snippet: Briefly, cells seeded in 96-well plates were loaded with the Ca 2+ -sensitive fluorescence indicator Fluo-4 (Life Technologies) and probenecid in Hanks' balanced salt solution, containing 1.3 m m CaCl 2 , 0.5 m m MgCl 2 ·6H 2 O, 0.4 m m MgSO 4 ·6H 2 O, 5.3 m m KCl, 0.4 m m KH 2 PO 4 , 4.2 m m NaHCO 3 , 137.9 m m NaCl, 0.3 m m Na 2 HPO 4 , 5.5 m m d -glucose, and 20 m m HEPES.

Techniques: Transfection, Microscopy, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: β 2 -Adrenergic Receptors Chaperone Trapped Bitter Taste Receptor 14 to the Cell Surface as a Heterodimer and Exert Unidirectional Desensitization of Taste Receptor Function *

doi: 10.1074/jbc.M116.722736

Figure Lengend Snippet: β 2 AR expression dynamically regulates TAS2R14 functional expression. HEK-293T cells were transfected with TAS2R14 + G α 16/G44, without or with β 2 AR ( A and B ). The [Ca 2+ ] i response to the TAS2R14 agonists DPD and FFA are increased when β 2 AR is co-transfected, consistent with the increased expression of TAS2R14 ( , A and B ). In 4 experiments, the [Ca 2+ ] i stimulation was increased by 2.4 ± 0.11 and 2.1 ± 0.12, respectively, when β 2 AR was co-expressed ( p < 0.01 versus absence of β 2 AR). In C , H292 cells, which endogenously express TAS2R14 and β 2 AR were transfected with β 2 AR shRNA (or sh-control) and treated with vehicle or the TAS2R14 agonist DPD. Knockdown of β 2 AR by β 2 AR shRNA resulted in decreased TAS2R14-mediated [Ca 2+ ] i signaling. In D and E , βAR knock-out mouse ASM cells (which express no detectable βAR) were challenged with TAS2R agonists and revealed >50% reduction in TAS2R-stimulated [Ca 2+ ] i . Results are from 4 representative experiments.

Article Snippet: Briefly, cells seeded in 96-well plates were loaded with the Ca 2+ -sensitive fluorescence indicator Fluo-4 (Life Technologies) and probenecid in Hanks' balanced salt solution, containing 1.3 m m CaCl 2 , 0.5 m m MgCl 2 ·6H 2 O, 0.4 m m MgSO 4 ·6H 2 O, 5.3 m m KCl, 0.4 m m KH 2 PO 4 , 4.2 m m NaHCO 3 , 137.9 m m NaCl, 0.3 m m Na 2 HPO 4 , 5.5 m m d -glucose, and 20 m m HEPES.

Techniques: Expressing, Functional Assay, Transfection, shRNA, Knock-Out

Journal: The Journal of Biological Chemistry

Article Title: β 2 -Adrenergic Receptors Chaperone Trapped Bitter Taste Receptor 14 to the Cell Surface as a Heterodimer and Exert Unidirectional Desensitization of Taste Receptor Function *

doi: 10.1074/jbc.M116.722736

Figure Lengend Snippet: Activation of β 2 AR within the β 2 AR:TAS2R14 heterodimer uncouples TAS2R14 signaling. HASM cells were treated with the β-agonist ISO (10 μ m ) for 5 min or 1 h, and then cells were challenged with the TAS2R14 agonist DPD (250 μ m ) with immediate quantitation of [Ca 2+ ] i release ( A ). Tracings are from a representative experiment performed in triplicate and the inset is mean ± S.E. of 4 experiments. *, p < 0.01 versus vehicle; +, p < 0.05 versus 5 min. In B , cells were treated with ISO or the cAMP stimulator forskolin (FSK, 10 μ m ) for 5 min and challenged with DPD as in A . Results shown are from a representative experiment of 4 performed, which showed no significant loss (10 ± 8%, p > 0.05) of DPD-stimulated [Ca 2+ ] i from forskolin treatment. In C cells were treated with the agonist ISO (10 μ m ), the neutral antagonist propranolol (10 μ m ), or the β 2 AR inverse agonist ICI118551 (10 μ m ) for 5 min and then challenged with 250 μ m of the TAS2R14 agonist DPD. There was no change in TAS2R14 function with ICI118551 or propranolol pretreatment. #, p < 0.01 versus vehicle, n = 4.

Article Snippet: Briefly, cells seeded in 96-well plates were loaded with the Ca 2+ -sensitive fluorescence indicator Fluo-4 (Life Technologies) and probenecid in Hanks' balanced salt solution, containing 1.3 m m CaCl 2 , 0.5 m m MgCl 2 ·6H 2 O, 0.4 m m MgSO 4 ·6H 2 O, 5.3 m m KCl, 0.4 m m KH 2 PO 4 , 4.2 m m NaHCO 3 , 137.9 m m NaCl, 0.3 m m Na 2 HPO 4 , 5.5 m m d -glucose, and 20 m m HEPES.

Techniques: Activation Assay, Quantitation Assay

Journal: The Journal of Biological Chemistry

Article Title: β 2 -Adrenergic Receptors Chaperone Trapped Bitter Taste Receptor 14 to the Cell Surface as a Heterodimer and Exert Unidirectional Desensitization of Taste Receptor Function *

doi: 10.1074/jbc.M116.722736

Figure Lengend Snippet: Effects of β 1 AR or β 2 AR activation on TAS2R14 signaling to [Ca 2+ ] i . H1299 cells were pretreated with carrier, the β 2 AR antagonist ICI118551 (10 μ m ), or the β 1 AR antagonist betaxolol (10 μ m ) for 10 min, and then cells were treated for 5 min with 10 μ m ISO. (The latter two conditions selectively activate β 1 AR and β 2 AR, respectively.) Desensitization of TAS2R14 signaling was observed with activation of either βAR subtype, and was greater when β 2 AR was activated compared with β 1 AR. *, p < 0.01 versus carrier; #, p < 0.01 + ICI118551 versus + betaxolol. Results are from 4 experiments.

Article Snippet: Briefly, cells seeded in 96-well plates were loaded with the Ca 2+ -sensitive fluorescence indicator Fluo-4 (Life Technologies) and probenecid in Hanks' balanced salt solution, containing 1.3 m m CaCl 2 , 0.5 m m MgCl 2 ·6H 2 O, 0.4 m m MgSO 4 ·6H 2 O, 5.3 m m KCl, 0.4 m m KH 2 PO 4 , 4.2 m m NaHCO 3 , 137.9 m m NaCl, 0.3 m m Na 2 HPO 4 , 5.5 m m d -glucose, and 20 m m HEPES.

Techniques: Activation Assay