To determine whether circulating factors may contribute to differences in cerebrovascular endothelial function between groups, we performed ex vivo experiments whereby human brain endothelial cells (HBECs) were treated with serum from the primary cohort. HBECs (ATCC Cat# CRL‐3245, RRID:CVCL_4D10) were plated in 96‐well culture plates and incubated under standard conditions (37°C, 5% CO2) for 2 h in basal media supplemented with 10% subject serum. After serum exposure, cells were co‐incubated with the fluorescent probes CellROX Deep Red (Thermo Fisher Scientific, Cat# C10422) to detect ROS production and DAR‐4 M‐AM (Sigma‐Aldrich, Cat# 251765) to detect NO production (Craighead et al., 2021 (link); Rossman et al., 2021 (link)). Cells were imaged before and 6 minutes after addition of 100 μM acetylcholine to stimulate NO production. Analysis was done with Celleste Image Analysis Software (Cellesta, v5, RRID:SCR_022692). ROS production was normalized to signal area and NO production was quantified as the fold change of post vs. pre‐acetylcholine stimulation.
Dar 4m am
Manufactured by Merck Group
Sourced in Austria
The DAR-4M-AM is a laboratory instrument designed for accurate and precise liquid handling tasks. It features a digital display, adjustable volume settings, and is suitable for use with a wide range of sample volumes. The core function of this product is to enable the controlled and repeatable transfer of liquids in laboratory environments.
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5 protocols using dar 4m am
1
Evaluating Cerebrovascular Endothelial Function
2
Quantifying Astrocytic Nitric Oxide
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Optical recordings of NO production in cultured astrocytes were performed using an inverted epifluorescence Olympus microscope, equipped with a cooled CCD camera (Clara model; Andor). The cells were loaded with the NO sensitive fluorescent probe DAR-4M-AM (Sigma; 10 µM, 30 min incubation at room temperature) or transduced to express the genetically encoded NO sensor geNOp. Recordings were performed in a custom-made flow-through imaging chamber at ~32°C in aCSF saturated with 95% O2 / 5% CO2 (pH 7.4). The rate of chamber perfusion with aCSF was 4 ml min-1. DAR-4M fluorescence was excited by using a Xenon arc lamp and an Optoscan Monochromator (Cairn Research) at 560/10 and the fluorescence emission was recorded at 590 nm. geNOp fluorescence was excited at 488/10 nm and the fluorescence emission was recorded at 535 nm.
Hypoxic conditions in vitro were induced by the displacement of oxygen in the medium by argon. In all experiments in cell cultures and organotypic slices the hypoxic challenge was applied for 5-15 min. A representative profile of PO2 changes in the recording chamber during argon displacement is illustrated byFigure 2F . All test drugs were applied ~10 min before the hypoxic challenge. Imaging data were collected and analyzed using Andor iQ3 software (Andor).
Hypoxic conditions in vitro were induced by the displacement of oxygen in the medium by argon. In all experiments in cell cultures and organotypic slices the hypoxic challenge was applied for 5-15 min. A representative profile of PO2 changes in the recording chamber during argon displacement is illustrated by
3
NO Sensing in Endothelial Cells
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After blocking and surface molecule staining (CD45 and CD31), cells were washed and incubated with the NO‐sensing fluorescent probe diaminorhodamine‐4M acetoxymethyl ester (DAR‐4M AM) (5 μm; Sigma) in PBS for 30 minutes at 37ºC. Finally, cells were fixed and analyzed with a FACSCanto II cytometer. Cells that were not incubated with the probe were used as negative controls for fluorescence. Two groups of cells could be distinguished according to the fluorescence levels at Em 580 nm. The fold change in the DAR‐4M AM mean fluorescence intensity (MFI) for ECs from WT mice and PSGL‐1−/− mice was calculated as the ratio of the MFI obtained in each mouse in relation to the average MFI for all WT mice analyzed.
4
Quantifying Endogenous Nitric Oxide
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To estimate endogenous levels of NO, unfixed pAVICs were incubated with 10 μM DAR-4M AM (Sigma-Aldrich, #251765) for 15 min at room temperature, followed by washing with phosphate-buffered saline (PBS). Nuclei were stained with DAPI. Images were captured using an Olympus IX51 microscope attached with an Olympus DP72 camera.
5
Measuring Astrocytic Nitric Oxide Dynamics
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Optical recordings of changes in NO production in cultured astrocytes were performed using an inverted epifluorescence Olympus microscope, equipped with a cooled CCD camera (Clara, Andor, Oxford Instruments). The cells were loaded with NO sensitive fluorescent probe DAR-4M-AM (Sigma; 10 µM for 30 min incubation at room temperature) or transduced to express genetically encoded NO sensor geNOp (NGFI; Austria). After incubation with the dye the cultures were washed three times with aCSF prior to the experiment. Recordings were performed in a custom-made flow-through imaging chamber at ~32°C in aCSF saturated with 95% O2 / 5% CO2 (pH 7.4). The rate of chamber perfusion with aCSF was 4 ml min -1 . To record changes in cytosolic [NO], DAR-4M or geNOp fluorescence were excited by using a Xenon arch lamp and a Optoscan Monocromator (Cairn Research) at 560/10 and 488/10 nm and the florescence emission was recorded at 590 and 535 nm, respectively. Hypoxic conditions in vitro were induced by displacement of oxygen in the medium with argon.
All test drugs were applied ~10 min before the hypoxic challenge. Imaging data were collected and analyzed using Andor iQ3 software (Andor, Belfast, UK). All data presented were obtained from at least 6 separate experiments.
All test drugs were applied ~10 min before the hypoxic challenge. Imaging data were collected and analyzed using Andor iQ3 software (Andor, Belfast, UK). All data presented were obtained from at least 6 separate experiments.
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