Live calcium ion staining and imaging was carried out to visualize the increase in intracellular calcium signaling in the cells as they get exposed to the piezoelectric surface charges of the PLLA films under US. For this assay, hADSCs were seeded on the films and allowed to attach overnight. Following this, the cells were stained with a Cal-520 AM dye (from AbCam) to visualize the calcium ion concentration in the culture. After the staining was completed following the manufacturer’s protocol for the dye, the films were transferred to glass bottom plates for imaging. Imaging was done using a fluorescent microscope (Leica) and each sample was imaged while being treated with US as well as without any US treatment to visualize the changes in calcium ion concentration. These changes were then quantified using ImageJ.
Cal 520am dye
Manufactured by Abcam
Cal-520 AM dye is a fluorescent calcium indicator that can be used for monitoring changes in intracellular calcium levels. It is a cell-permeable compound that becomes fluorescent upon binding to calcium ions.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescent microscope, by Leica (1 mentions)
Ryanodine, by Merck Group (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Ca free keratinocyte sfm medium k sfm, by Thermo Fisher Scientific (1 mentions)
Amg9810, by Merck Group (1 mentions)
Bapta am, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Thapsigargin, by Abcam (1 mentions)
Sp5 confocal microscope, by Leica (1 mentions)
4 protocols using cal 520am dye
1
Piezoelectric PLLA Films Induce Calcium Signaling
2
Astrocytes Calcium Imaging with Sulfatides
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Astrocytes were seeded in a 24 well-plate and grown for 24 h in supplemented DMEM/FBS until 80% confluent. Cells were then serum-starved for 4 h prior to treatment with sulfatides (5 µM, 10 µM, 20 µM, 50 µM, and 100 µM) with or without 100 nM Olaparib for 24 h. After treatment, cells were incubated with 3 μM Cal-520AM dye (Abcam, ab171868) in Hank's balanced salt solution (HBSS; no phenol red) supplemented with 10 mM glucose and 25 mM HEPES for 90 min at 37 °C, followed by 30 min at room temperature. Cells were protected from bright light at all times. Cells were then incubated in HBSS minus Ca2+ and Mg2+ (HBSS−/−). Time-lapse Ca2+ imaging was performed at a rate of 0.99 frames per second. Following a 20 s baseline, cells were stimulated with 300 μM ATP for 240 s. Intracellular Ca2+ imaging experiments were performed with HBSS minus Ca2+ and Mg2+ (HBSS−/−). Images were acquired using a motorised epifluorescent microscope and a ×20 magnification objective.
3
Calcium Signaling in Keratinocyte Culture
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Active reagents and the concentrations used were selected from previous TPMD literature7 (link),35 (link)–37 (link) and from the TRP channel literature38 (link). AMG 9810, AMTB, ryanodine, BAPTA-AM, 18-α glycyrrhetinic acid (18α-GA), apyrase and DAPI were purchased from Sigma-Aldrich, St. Louis, MO; thapsigargin, BCTC, and Cal-520-AM dye were purchased from Abcam, Cambridge, United Kingdom, Focus Biomolecules, Plymouth Meeting, PA, and AAT Bioquest, Sunnyvale, CA, respectively. Polyclonal connexin 43 antibody was purchased from Cell Signaling, Danvers, MA; Alexa Fluor 488 was purchased from Fisher, Pittsburgh, PA. Ca++-free Keratinocyte-SFM medium (K-SFM) was purchased from ThermoFisher Scientific (Cat. No.: 10725–018), Waltham, MA.
4
Live-cell Calcium Imaging of Astrocytes
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Astrocytes were seeded onto 25 mm tissue culture plastic coverslips (Sarstedt, #83.1840) in a 6 well-plate and grown for 72 h in supplemented DMEM/F12 until 70% confluent. Cells were then serum-starved for 3 h prior to treatment with or without 100 ng/mL LPS for 24 h. After treatment, cells were incubated with 3 μM Cal-520AM dye (Abcam, ab171868) in Hank's balanced salt solution (HBSS; no phenol red) supplemented with 10 mM glucose and 25 mM HEPES for 90 min at 37 o C, followed by 30 min at 22 o C. Cells were protected from bright light at all times. Coverslips were then placed into the Attofluor TM acquisition chamber (Invitrogen) with supplemented HBSS or HBSS minus Ca 2+ and Mg 2+ (HBSS -/-). Time-lapse Ca 2+ imaging was performed at a rate of 0.77 frames per second. Following a 30 second baseline, cells were stimulated with either HBSS or 10 μM Yoda1 for 240 seconds and stimulated further with 50 μM ATP for 180 seconds. Extracellular Ca 2+ imaging experiments were performed with standard HBSS and intracellular Ca 2+ imaging experiments were performed with HBSS minus Ca 2+ and Mg 2+ (HBSS -/-). Images were acquired using a Leica SP5 confocal microscope and a 20× magnification objective.
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