Culture of brain capillary pericytes for cytosolic calcium measurements and calcium imaging studies
Research output: Contribution to journal › Journal article › peer-review
Standard
Culture of brain capillary pericytes for cytosolic calcium measurements and calcium imaging studies. / Hørlyck, Sofie; Helms, Hans Christian Cederberg; Brodin, Birger.
In: Journal of Visualized Experiments, Vol. 159, e61253, 05.2020, p. 1-9.Research output: Contribution to journal › Journal article › peer-review
Harvard
APA
Vancouver
Author
Bibtex
}
RIS
TY - JOUR
T1 - Culture of brain capillary pericytes for cytosolic calcium measurements and calcium imaging studies
AU - Hørlyck, Sofie
AU - Helms, Hans Christian Cederberg
AU - Brodin, Birger
PY - 2020/5
Y1 - 2020/5
N2 - Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte function is not fully known. Pericytes have been suggested to be involved in capillary development, regulation of endothelial barrier tightness and trancytosis activity, regulation of capillary tone and to play crucial roles in certain brain pathologies. Pericytes are challenging to investigate in the intact brain due to the difficulties in visualizing processes in the brain parenchyma, as well as the close proximity to the other cells of the NVU. The present protocol describes a method for isolation and culture of primary bovine brain capillary pericytes and their following usage in calcium imaging studies, where effects of agonists involved in brain signaling and pathologies can be investigated. Cortical capillary fragments are allowed to attach to the bottom of culture flasks and, after 6 days, endothelial cells and pericytes have grown out from the capillary fragments. The endothelial cells are removed by gentle trypsinization and pericytes are cultured for 5 additional days before passaging. Isolated pericytes are seeded in 96-well culture plates and loaded with the calcium indicator dye (Fura-2 acetoxymethyl (AM)) to allow for measurements of intracellular calcium levels in a plate reader setup. Alternatively, pericytes are seeded on coverslips and mounted in cell chambers. Following loading with the calcium indicator (Cal-520 AM), calcium live-imaging can be performed using confocal microscopy at an excitation wavelength of 488 nm and emission wavelength of 510-520 nm. The method described here has been used to obtain the first intracellular calcium measurements from primary brain capillary pericytes, demonstrating that pericytes are stimulated via ATP and are able to contract in vitro.
AB - Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte function is not fully known. Pericytes have been suggested to be involved in capillary development, regulation of endothelial barrier tightness and trancytosis activity, regulation of capillary tone and to play crucial roles in certain brain pathologies. Pericytes are challenging to investigate in the intact brain due to the difficulties in visualizing processes in the brain parenchyma, as well as the close proximity to the other cells of the NVU. The present protocol describes a method for isolation and culture of primary bovine brain capillary pericytes and their following usage in calcium imaging studies, where effects of agonists involved in brain signaling and pathologies can be investigated. Cortical capillary fragments are allowed to attach to the bottom of culture flasks and, after 6 days, endothelial cells and pericytes have grown out from the capillary fragments. The endothelial cells are removed by gentle trypsinization and pericytes are cultured for 5 additional days before passaging. Isolated pericytes are seeded in 96-well culture plates and loaded with the calcium indicator dye (Fura-2 acetoxymethyl (AM)) to allow for measurements of intracellular calcium levels in a plate reader setup. Alternatively, pericytes are seeded on coverslips and mounted in cell chambers. Following loading with the calcium indicator (Cal-520 AM), calcium live-imaging can be performed using confocal microscopy at an excitation wavelength of 488 nm and emission wavelength of 510-520 nm. The method described here has been used to obtain the first intracellular calcium measurements from primary brain capillary pericytes, demonstrating that pericytes are stimulated via ATP and are able to contract in vitro.
KW - ATP
KW - Ca-signaling
KW - Cal-520 AM
KW - Cell culture
KW - Confocal laser scanning microscopy
KW - Fura-2 AM
KW - Issue 159
KW - Neuroscience
KW - Pericyte
KW - Plate reader
U2 - 10.3791/61253
DO - 10.3791/61253
M3 - Journal article
C2 - 32538913
AN - SCOPUS:85086226734
VL - 159
SP - 1
EP - 9
JO - Journal of Visualized Experiments
JF - Journal of Visualized Experiments
SN - 1940-087X
M1 - e61253
ER -
ID: 243154153