PMID- 32538913
OWN - NLM
STAT- MEDLINE
DCOM- 20201023
LR  - 20201023
IS  - 1940-087X (Electronic)
IS  - 1940-087X (Linking)
IP  - 159
DP  - 2020 May 27
TI  - Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and 
      Calcium Imaging Studies.
LID - 10.3791/61253 [doi]
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.
FAU - Horlyck, Sofie
AU  - Horlyck S
AD  - Department of Pharmacy, The Faculty of Health and Medical Sciences, University of 
      Copenhagen.
FAU - Helms, Hans Christian Cederberg
AU  - Helms HCC
AD  - Department of Pharmacy, The Faculty of Health and Medical Sciences, University of 
      Copenhagen.
FAU - Brodin, Birger
AU  - Brodin B
AD  - Department of Pharmacy, The Faculty of Health and Medical Sciences, University of 
      Copenhagen; birger.brodin@sund.ku.dk.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Video-Audio Media
DEP - 20200527
PL  - United States
TA  - J Vis Exp
JT  - Journal of visualized experiments : JoVE
JID - 101313252
RN  - SY7Q814VUP (Calcium)
SB  - IM
MH  - Animals
MH  - Brain
MH  - Calcium/*analysis
MH  - Capillaries/*cytology
MH  - Cattle
MH  - *Cell Culture Techniques
MH  - Cell Separation
MH  - Cytosol/chemistry
MH  - Microscopy, Confocal
MH  - Pericytes/*cytology
EDAT- 2020/06/17 06:00
MHDA- 2020/10/24 06:00
CRDT- 2020/06/16 06:00
PHST- 2020/06/16 06:00 [entrez]
PHST- 2020/06/17 06:00 [pubmed]
PHST- 2020/10/24 06:00 [medline]
AID - 10.3791/61253 [doi]
PST - epublish
SO  - J Vis Exp. 2020 May 27;(159). doi: 10.3791/61253.