Culture of brain capillary pericytes for cytosolic calcium measurements and calcium imaging studies

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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 journalJournal articlepeer-review

Harvard

Hørlyck, S, Helms, HCC & Brodin, B 2020, 'Culture of brain capillary pericytes for cytosolic calcium measurements and calcium imaging studies', Journal of Visualized Experiments, vol. 159, e61253, pp. 1-9. https://doi.org/10.3791/61253

APA

Hørlyck, S., Helms, H. C. C., & Brodin, B. (2020). Culture of brain capillary pericytes for cytosolic calcium measurements and calcium imaging studies. Journal of Visualized Experiments, 159, 1-9. [e61253]. https://doi.org/10.3791/61253

Vancouver

Hørlyck S, Helms HCC, Brodin B. Culture of brain capillary pericytes for cytosolic calcium measurements and calcium imaging studies. Journal of Visualized Experiments. 2020 May;159:1-9. e61253. https://doi.org/10.3791/61253

Author

Hørlyck, Sofie ; Helms, Hans Christian Cederberg ; Brodin, Birger. / Culture of brain capillary pericytes for cytosolic calcium measurements and calcium imaging studies. In: Journal of Visualized Experiments. 2020 ; Vol. 159. pp. 1-9.

Bibtex

@article{62591890004243e890cd320290becae6,
title = "Culture of brain capillary pericytes for cytosolic calcium measurements and calcium imaging studies",
abstract = "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.",
keywords = "ATP, Ca-signaling, Cal-520 AM, Cell culture, Confocal laser scanning microscopy, Fura-2 AM, Issue 159, Neuroscience, Pericyte, Plate reader",
author = "Sofie H{\o}rlyck and Helms, {Hans Christian Cederberg} and Birger Brodin",
year = "2020",
month = may,
doi = "10.3791/61253",
language = "English",
volume = "159",
pages = "1--9",
journal = "Journal of Visualized Experiments",
issn = "1940-087X",
publisher = "Journal of Visualized Experiments",

}

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