The living CMs on the device substrate were rinsed with 1 ml of Hepes-buffered Tyrode’s solution (Thermo Fisher Scientific) and stained with Fluo-8 AM (5 μM; AAT Bioquest). The cells were then incubated at 37°C for 1 hour. The device was then rinsed with Tyrode’s solution three times. The calcium transient signal (fig. S10) was recorded with a ZEISS Axio Examiner microscope system equipped with a CCD camera (Axiocam 702 Mono Camera) and ZEN Blue software at 12 fps. The recorded signal was analyzed by ImageJ.
Hepes buffered tyrode s solution
Manufactured by Thermo Fisher Scientific
HEPES-buffered Tyrode's solution is a balanced salt solution that maintains the pH and osmotic pressure of cell cultures. It is commonly used as a physiological buffer in various cell culture applications.
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Lab products found in correlation
6 protocols using hepes buffered tyrode s solution
1
Cell calcium transient analysis
2
Fluorescent Calcium Imaging of CMT
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The mesh-innervated CMT in the chamber was rinsed with 1 ml of Hepes-buffered Tyrode’s solution (Thermo Fisher Scientific) and stained with Fluo-8 AM (5 mM, AAT Bioquest). The CMT was then incubated for 1 h at 37 °C. Subsequently, the system was rinsed with Tyrode’s solution three times. The Ca2+ transient signal was recorded with a confocal microscope (Nikon A1R). Electrical recordings were performed simultaneously. The recorded signals were analyzed by ImageJ.
3
Calcium Imaging of Neurons and Astrocytes
4
Spectral Imaging of FRET Biosensors
5
Calcium Imaging of Neurons and Astrocytes
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Calcium imaging was performed on cells seeded on a 30 mm glass bottom dish (TED PELLA) coated with Matrigel. Neurons were incubated with 1 µM Fluo-4-AM (Invitrogen) and 0.02% Pluronic™ F-127 in DMEM for 30 min at 37 °C. To reduce dye associated cytotoxicity, astrocytes were incubated with 0.5 µM Fluo-4-AM (Invitrogen) and 0.02% Pluronic™ F–127 in DMEM for 15 min at 37 °C. Cells were then washed once with HEPES-buffered Tyrode’s solution (Alfa Aesar), and allowed to de-esterify at RT in the dark for 20 min prior to imaging on a Nikon Ti2-Eclipse inverted microscope. Fluorescent time-lapse images for both neurons and astrocytes were acquired (20x objective) using an 80 ms exposure time at one frame per second for 300 s. Calcium spike traces were generated by quantifying the mean pixel intensity of manually identified regions of interest using Fiji [30 (link)].
6
Calcium Imaging of Astrocytes
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For calcium imaging analysis, astrocytes
were seeded at a density of 2 × 105 in a glass 40
mm dish. Cells were incubated with 2 μM Fluo-4 AM (ThermoFisher)
and 0.02% Pluronic F-127 in DMEM for 15 min at 37 °C. After one
wash in HEPES-buffered Tyrode’s solution (Alfa Aesar), the
dye was allowed to de-esterify by incubating the cells at room temperature
for 20 min prior to imaging on a Zeiss AxioObserver Z1. Fluorescent
time-lapse images were acquired (20× objective) at 1 s intervals
for 360 s. Calcium spike traces were generated by quantifying the
mean pixel intensity of manually identified regions of interest using
the ImageJ software. For astrocytes cultured on microcarriers, cells
differentiated for at least 45 days were replated at a density of
1.4 × 106 cells onto VDP-coated glass 40 mm dishes
and cultured for 2–4 days prior to staining and imaging.
were seeded at a density of 2 × 105 in a glass 40
mm dish. Cells were incubated with 2 μM Fluo-4 AM (ThermoFisher)
and 0.02% Pluronic F-127 in DMEM for 15 min at 37 °C. After one
wash in HEPES-buffered Tyrode’s solution (Alfa Aesar), the
dye was allowed to de-esterify by incubating the cells at room temperature
for 20 min prior to imaging on a Zeiss AxioObserver Z1. Fluorescent
time-lapse images were acquired (20× objective) at 1 s intervals
for 360 s. Calcium spike traces were generated by quantifying the
mean pixel intensity of manually identified regions of interest using
the ImageJ software. For astrocytes cultured on microcarriers, cells
differentiated for at least 45 days were replated at a density of
1.4 × 106 cells onto VDP-coated glass 40 mm dishes
and cultured for 2–4 days prior to staining and imaging.
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