All experiments were performed in dark conditions. The cells were loaded with a Rhod-2 AM dye (1 μM) (Kd = 570 nM, λEx = 552 nm and λEm = 581 nm) (Invitrogen, CA, USA) for 45min. The cells were then washed with PBS twice before being incubated with Hanks buffer, and treated with the appropriate NPs concentration. All Ca2+ signaling experiments were carried out in a thermo-regulated state at 37°C mounted on a Nikon microscope (Nikon Eclipse TE2000- U, Tokyo, Japan) (Chen et al., 2008 ; Chen et al., 2011 (link); Chen et al., 2012 (link); Tsai et al., 2017 ).
Rhod2 am dye
Manufactured by Thermo Fisher Scientific
Sourced in United States
Rhod2-AM is a fluorescent dye that is commonly used for the measurement of intracellular calcium levels. It is a cell-permeable derivative of the calcium indicator dye rhodamine-2. When Rhod2-AM enters a cell, it is hydrolyzed by intracellular esterases, releasing the active Rhod2 dye which then binds to calcium ions, resulting in increased fluorescence. This property makes Rhod2-AM a useful tool for studying calcium-dependent cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Pluronic f27, by Thermo Fisher Scientific (3 mentions)
Fluo 4 am, by Thermo Fisher Scientific (3 mentions)
Axiovert s100 microscope, by Zeiss (2 mentions)
Oxotremorine m, by Bio-Techne (2 mentions)
Metamorph, by GE Healthcare (2 mentions)
Eclipse te2000 u, by Nikon (1 mentions)
Plan apo vc 20 objective, by Nikon (1 mentions)
Oxotremorine m, by Santa Cruz Biotechnology (1 mentions)
Nit 1 cells, by American Type Culture Collection (1 mentions)
Ham s f12 medium, by Merck Group (1 mentions)
Fluo 3 am, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Merck Group (1 mentions)
Plan neofluar objective, by Zeiss (1 mentions)
6 protocols using rhod2 am dye
1
Calcium Signaling Assay for Nanoparticles
2
Measurement of Ca2+ Dynamics in SH-SY5Y Cells
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Ca2+ measurements were performed essentially as described previously [20 (link), 78 (link), 79 (link)]. SH-SY5Y cells were loaded with 2 μM Fluo4-AM and/or Rhod2-AM dye (Invitrogen) in external solution (145 mM NaCl, 2 mM KCl, 5 mM NaHCO3, 1 mM MgCl2, 2.5 mM CaCl2, 10 mM glucose, 10 mM Na-HEPES, pH 7.25) containing 0.02% Pluronic-F27 (Invitrogen) for 15 min at 37 °C, followed by washing in external solution for 15 min at 37 °C. Fluo4 and Rhod2 fluorescence were timelapse recorded (1 s intervals) at 37 °C using either an Axiovert S100 microscope (Zeiss) driven by MetaMorph (Molecular Dynamics) and equipped with GFP (Fluo4) and DsRed (Rhod2) filtersets (Chroma Technology), a 40× Plan-Neofluar 1.3NA objective (Zeiss), and a Photometrics Cascade-II 512B36 EMCCD camera or a Nikon Ti-E microscope using a CFI Plan Apo VC 20× objective and Nikon Andor Neo sCMOD high-resolution camera and appropriate filter sets. The cells were kept under constant perfusion with external solution (0.5 ml/min). Inositol 1,4,5-trisphosphate (IP3) receptor-mediated Ca2+ release from ER stores was triggered by application of 100 μM Oxotremorine-M for 2 min. Ca2+ levels were calculated as relative Rhod2 or Fluo4 fluorescence compared to baseline fluorescence (F/F0) at the start of the measurement. Oxotremorine-M was from Santa Cruz Biotechnology and was dissolved in water.
3
Cytosolic and Mitochondrial Calcium Dynamics in NIT-1 Cells
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NIT1 cells (ATCC) were cultured in Ham’s F12 medium (Sigma-Aldrich) supplemented with 15% FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml of streptomycin (All reagents from GIBCO, USA). For experiments, NIT-1 cells were seeded at a density of 40,000 cells/well in 96-well plates and incubated for 72 h. Cells were loaded with Fluo-3 AM (Invitrogen, 2.5 μM) dye for cytosolic calcium measurement or Rhod-2 AM dye (Invitrogen, 4 μM) for mitochondrial calcium measurement for 60 min and washed twice with HEPES buffered saline solution (HBSS) to remove unbound dye. Following a basal measurement, cells were treated with low glucose (2.8 mM), high glucose (16.7 mM) or high glucose with 1 μM of CNX-011-67 and Ca2+ levels were measured for 4 min at 6-s intervals using a Biotek Synergy 2 fluorimeter with resulting flux being represented as arbitrary fluorescence units (AFU) [40 (link)]. Calcium levels represented refer to net response with background fluorescence corrections. There were no oscillations observed and the response was sustained for nearly 12 minutes.
4
Monitoring Mitochondrial Calcium Dynamics
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To monitor the mitochondrial matrix calcium level ([Ca2+]m), cells were loaded with 0.2 μM cell-permeant mitochondrial specific fluorescent Ca2+ indicator Rhod-2 AM dye (Invitrogen, USA) at 4 °C for 1 h, followed by incubation at 37 °C for 30 min as previously described [38 (link)]. The Rhod-2 imaging was performed with an excitation wavelength of 561 nm, and 3D imaging of cells was performed consisting of 120 stacks that span 5 μm using a 63X oil objective. To analyze [Ca2+]m level, we performed a 2D compression of all z-stacks in 5 μm, and regions of interest (n = 10) were drawn manually in the 30 cells corresponding to each treatment (quantification of mitochondrial calcium accumulation). Further, the average Rhod-2 fluorescence intensity in each cell was quantified using LASX software. Rhod-2 staining and imaging were performed to monitor mitochondrial Ca2+ for control cells, nifedipine treated cells, CoCl2 treated cells, and CoCl2 treated cells, followed by nifedipine treatment.
5
Measuring Cytosolic and Mitochondrial Ca2+ Levels
6
Measuring Cytosolic and Mitochondrial Ca2+ Levels
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Cytosolic and mitochondrial Ca2+ levels were measured following
IP3R-mediated release from ER stores in HEK293 cells20 (link). To
do so, cells transfected with M3R and either empty vector control plasmid or TDP-43 plasmids were loaded with either
2 μM Fluo4-AM or Rhod2-AM dye (Invitrogen) in external solution
(145 mM NaCl,
2 mM KCl,
5 mM NaHCO3, 1 mM MgCl2, 2.5 mM
CaCl2,
10 mM glucose and
10 mM Na-HEPES pH
7.25) containing 0.02% Pluronic-F27 (Invitrogen) for 15 min at
37 °C, followed by washing in external solution for
15 min. Fluo4 and
Rhod2 fluorescence were
timelapse recorded (1-s intervals) with MetaMorph (Molecular Dynamics) on an
Axiovert S100 microscope (Zeiss) equipped with appropriate filtersets (Chroma
Technology), a × 40/1.3NA Plan-Neofluar objective (Zeiss) and a
Photometrics Cascade-II 512B EMCCD. The cells were kept under constant perfusion
with external solution
(0.5 ml min−1).
IP3R-mediated Ca2+ release from ER stores was triggered by
application of 100 μM Oxotremorine-M (Tocris) for 2 min.
Ca2+ levels were calculated as relative Fluo4 or Rhod2 fluorescence compared with
baseline fluorescence at the start of the measurement.
IP3R-mediated release from ER stores in HEK293 cells20 (link). To
do so, cells transfected with M3R and either empty vector control plasmid or TDP-43 plasmids were loaded with either
2 μM Fluo4-AM or Rhod2-AM dye (Invitrogen) in external solution
(145 mM NaCl,
2 mM KCl,
5 mM NaHCO3, 1 mM MgCl2, 2.5 mM
CaCl2,
10 mM glucose and
10 mM Na-HEPES pH
7.25) containing 0.02% Pluronic-F27 (Invitrogen) for 15 min at
37 °C, followed by washing in external solution for
15 min. Fluo4 and
Rhod2 fluorescence were
timelapse recorded (1-s intervals) with MetaMorph (Molecular Dynamics) on an
Axiovert S100 microscope (Zeiss) equipped with appropriate filtersets (Chroma
Technology), a × 40/1.3NA Plan-Neofluar objective (Zeiss) and a
Photometrics Cascade-II 512B EMCCD. The cells were kept under constant perfusion
with external solution
(0.5 ml min−1).
IP3R-mediated Ca2+ release from ER stores was triggered by
application of 100 μM Oxotremorine-M (Tocris) for 2 min.
Ca2+ levels were calculated as relative Fluo4 or Rhod2 fluorescence compared with
baseline fluorescence at the start of the measurement.
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