rat brain endothelial cell basal medium  (Cell Applications Inc)

Journal: Cell reports

Article Title: Two-pore channel-2 and inositol trisphosphate receptors coordinate Ca 2+ signals between lysosomes and the endoplasmic reticulum

doi: 10.1016/j.celrep.2023.113628

Figure Lengend Snippet: (A‒C) Effect of TPC2-A1-N or TPC2-A1-P on histamine-induced Ca 2+ signals in HeLa cells loaded with Fura-2. (A) Time course showing the effect of co-stimulating cell populations in an automated manner with increasing concentrations of histamine and a fixed concentration of DMSO (0.1% [v/v]), TPC2-A1-N (N, 30 μM), or TPC2-A1-P (P, 60 μM). (B) Concentration-effect relationship for peak histamine responses in the presence of indicated reagents. (C) Effect of co-stimulating cells with increasing concentrations of TPC2-A1-N or TPC2-A1-P at a fixed concentration of histamine (0.8 μM, left, or 2.6 μM, right). Data are expressed as mean ± SEM from 3–4 biological replicates. (D and E) Effect of TPC2-A1-N or TPC2-A1-P on histamine-induced Ca 2+ signals in U2OS cells loaded with Fura-2. (D) Time course showing the effect of co-stimulating cell populations in an automated manner with increasing concentrations of histamine and a fixed concentration of DMSO (0.1% [v/v]) or TPC2-A1-N (10 μM). (E) Concentration-effect relationship for peak histamine responses in the presence of TPC2-A1-N (10 μM) or TPC2-A1-P (20 μM). Data are expressed as mean ± SEM from 3–4 biological replicates. (F and G) Effect of TPC2-A1-N on histamine-induced NO production in rat brain microvascular endothelial cells (RBMVECs) loaded with DAF 2A. (F) Time course data showing the effect of co-stimulating cells with increasing concentrations of histamine and a fixed concentration of DMSO (0.1% [v/v]) or TPC2-A1-N (N, 10 μM). Each trace is the fluorescence response of a single cell. The thicker trace is the average of the population. (G) Concentration-effect relationship for peak histamine responses in the presence of indicated reagents. Data were corrected for basal NO production in the absence of histamine. Data are expressed as mean ± SEM from 27–33 cells. ****p < 0.0001, n.s., not significant, two-way ANOVA followed by Bonferroni’s test. (H) Proposed model whereby local Ca 2+ signals stemming from the lysosome via TPC2 and the ER via IP 3 receptors form regional intermediaries that precede global Ca 2+ signals upon cellular stimulation. Created with BioRender.com .

Article Snippet: Rat brain microvascular endothelial cells (Cell Applications, Inc. (San Diego, CA, USA) were cultured in rat brain endothelial cell basal medium and rat brain endothelial cell growth supplement in flasks coated with attachment factor according to the manufacturer’s instructions (Cell Applications, Inc.) , For single cell epifluorescence imaging, HeLa and U2OS cells were plated onto round 13 mm diameter coverslips (Academy) coated with poly-L-lysine (20 μg/mL, Sigma).

Techniques: Concentration Assay, Fluorescence, Cell Stimulation

Journal: Cell reports

Article Title: Two-pore channel-2 and inositol trisphosphate receptors coordinate Ca 2+ signals between lysosomes and the endoplasmic reticulum

doi: 10.1016/j.celrep.2023.113628

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rat brain microvascular endothelial cells (Cell Applications, Inc. (San Diego, CA, USA) were cultured in rat brain endothelial cell basal medium and rat brain endothelial cell growth supplement in flasks coated with attachment factor according to the manufacturer’s instructions (Cell Applications, Inc.) , For single cell epifluorescence imaging, HeLa and U2OS cells were plated onto round 13 mm diameter coverslips (Academy) coated with poly-L-lysine (20 μg/mL, Sigma).

Techniques: Recombinant, CRISPR, Plasmid Preparation, Software

Journal: Brain research

Article Title: Mechanisms of modulation of brain microvascular endothelial cells function by thrombin

doi: 10.1016/j.brainres.2016.12.011

Figure Lengend Snippet: A, Distribution of F-actin (red), a component of cytoskeleton, and ZO-1 (green), a component of tight junctions, in RBMVEC in control cells, cells treated with thrombin (0.5 u/ml) or thrombin (0.5 u/ml) and FR-171113 (1 μM). Treatment with thrombin increased F-actin stress fiber formation, produced a reduction in ZO-1 staining, indicating cytoskeletal rearrangement and disruption of tight junctions; in addition, intercellular gaps, indicated by arrows, became visible in the endothelial monolayer. Pretreatment with the PAR1 antagonist prevented the changes produced by thrombin. Cellular nuclei were stained with DAPI. B, Thrombin increased the permeability of RBMVEC monolayers assessed using the FITC-dextran flux assay *P < 0.05 as compared to control.

Article Snippet: Cell Culture Rat brain microvascular endothelial cells (RBMVEC) from Cell Applications, Inc (San Diego, CA) were cultured in rat brain endothelial basal medium and endothelial growth supplements, according to the manufacturer’s instructions (Cell Applications, Inc), as previously described ( Altmann et al., 2015 ; Brailoiu et al., 2016 ).

Techniques: Control, Produced, Staining, Disruption, Permeability, Flux Assay