Retrograde Analysis of Calcium Signaling by CaMPARI2 Shows Cytosolic Calcium in Chondrocytes Is Unaffected by Parabolic Flights
Abstract
:1. Introduction
1.1. Genetically Encoded Ca2+ Indicators
1.2. CaMPARI
1.3. Chondrocytes and Histamine
2. Materials and Methods
2.1. Vector Construction
2.2. Cell Culture and Treatment
2.3. Chondrocyte Transfection
2.4. Parabolic Flight Experimental Design
2.5. Parabolic Flight Hardware
2.6. Thermal Management
2.7. Before the Flight
2.8. During the Flight
2.9. After the Flight
2.10. Image Analysis and Statistics
3. Results
3.1. Selection of CaMPARI2-Constructs
3.2. Gravi-Elicited Ca2+ Elevation Changes during a Parabolic Flight
3.3. Construct and Flight-Specific Conversion Rate
3.4. Inhibitor Specific Conversion Rate
3.5. Z’-Factor Analysis
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Drug | Concentration | Solvent | Action | Reference |
---|---|---|---|---|
Untreated | - | - | - | |
DMSO | 0.1% | - | DMSO control | |
Gadolinium (III) chloride (Gd3+) | 10 µM | H2O | Blocker of extracellular calcium (Ca2+) entry | [38,40,41,46,49,59,60,61] |
Thapsigargin | 1 µM | 0.1% DMSO | Blocker of SERCA (sarco/endoplasmic reticulum Ca2+-ATPase) | [47] |
Flunarizine | 50 µM | 0.1% DMSO | Blocker of Ca2+ entry and histamine H1 | [62,63] |
Ruthenium red | 10 µM | H2O | Blocker of CatSper1, KCNK3, RyR1, RyR2, RyR3, TRPM6, TRPM8, TRPV1, TRPV2, TRPV3, TRPV4, TRPV5, TRPV6, TRPA1, CALHM1, TRPP3, PIEZO | [64] |
GSK2193874 | 10 µM | 0.1% DMSO | Selective blocker of TRPV4 | [65] |
BAPTA-AM | 10 µM | 0.1% DMSO | Cell-permeable Ca2+-chelator | [66] |
Metric | Definition |
---|---|
Conversion rate | The proportion of the green fluorescent cells that also display a red fluorescence (photoconversion) |
Red objects mean fluorescence intensity | The mean red intensity of all cells |
Merged objects red mean intensity | The mean red intensity of all cells displaying green and red fluorescence |
Red-integrated intensity per well | The red intensity per well divided by the number of cells per well that display green and red fluorescence |
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Hammer, A.; Cerretti, G.; Ricciardi, D.A.; Schiffmann, D.; Maranda, S.; Kummer, R.; Zumbühl, C.; Rattenbacher-Kiser, K.F.; von Arx, S.; Ammann, S.; et al. Retrograde Analysis of Calcium Signaling by CaMPARI2 Shows Cytosolic Calcium in Chondrocytes Is Unaffected by Parabolic Flights. Biomedicines 2022, 10, 138. https://doi.org/10.3390/biomedicines10010138
Hammer A, Cerretti G, Ricciardi DA, Schiffmann D, Maranda S, Kummer R, Zumbühl C, Rattenbacher-Kiser KF, von Arx S, Ammann S, et al. Retrograde Analysis of Calcium Signaling by CaMPARI2 Shows Cytosolic Calcium in Chondrocytes Is Unaffected by Parabolic Flights. Biomedicines. 2022; 10(1):138. https://doi.org/10.3390/biomedicines10010138
Chicago/Turabian StyleHammer, Andreas, Geraldine Cerretti, Dario A. Ricciardi, David Schiffmann, Simon Maranda, Raphael Kummer, Christoph Zumbühl, Karin F. Rattenbacher-Kiser, Silvan von Arx, Sebastian Ammann, and et al. 2022. "Retrograde Analysis of Calcium Signaling by CaMPARI2 Shows Cytosolic Calcium in Chondrocytes Is Unaffected by Parabolic Flights" Biomedicines 10, no. 1: 138. https://doi.org/10.3390/biomedicines10010138