eoe sub articles

EoE Sub Articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 
1. Preparation of the materials
<ol>
	<li>Magnetic-activated cell sorting (MACS) buffer: Prepare 2 mM ethylene diamine tetraacetic acid (EDTA) and 0.5% bovine serum albumin (BSA) (Table of Materials) in PBS-/-.</li>
	<li>Collagenase IV stock solution (20 U/&#956;L): Resuspend the powder in PBS+/+ (Table of ...

isolating immune cells from mouse choroid plexuses

Source: Dominguez-Belloso, et al. <a href="https://app.jove.com/v/63487">Isolation and Characterization of the Immune Cells from Micro-dissected Mouse Choroid Plexuses.</a> J. Vis. Exp. (2022)
This video demonstrates the procedure of isolating immune cells from mouse choroid plexuses. This procedure is used in neuroimmunology research to understand how immune cells in the choroid plexuses impact brain health and various neurological diseases.

Isolating Immune Cells from Mouse Choroid Plexuses

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 
1. ...

Position a mouse&#39;s brain dorsal side up.
Under a microscope, separate the cerebral hemispheres. Pull away the cortex and then the hippocampus to expose the lateral ventricles and the third ventricle.
Collect two lateral choroid plexuses. These are immune-cell-rich brain structures that line the lateral ventricles like a long veil.
Collect the third ventricle choroid plexus, which is a short structure with a granular surface.
Detach the cerebellum to expose the fourth ventricle.
Collect the fourth ventricle choroid plexus, which is a long globular structure with a granular surface.
Place all the choroid plexuses in a tube with buffered saline to maintain tissue integrity.
Add a digestive enzyme solution and incubate with agitation.
The enzyme breaks down the extracellular matrix and loosens the cells.
Add a suitable buffer to inactivate the enzyme.
Centrifuge to settle the cells. Discard the supernatant and resuspend the cells in a fresh buffer for further analysis.

isolating-immune-cells-from-mouse-choroid-plexuses

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Primary Neuronal Culture

Characterization of Immune Cells in Human Adipose Tissue by Using Flow Cytometry

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the development of obesity-associated complications such as type 2 diabetes. To quantitatively and qualitatively investigate the immune cell subsets in human AT deposits, we have developed a flow cytometry approach. The stromal vascular fraction (SVF), containing the immune cells, is isolated from subcutaneous and visceral AT biopsies by collagenase digestion. Adipocytes are removed after centrifugation. The SVF cells are stained for multiple membrane-bound markers selected to differentiate between immune cell subsets and analyzed using flow cytometry. As a result of this approach, pro- and anti-inflammatory macrophage subsets, dendritic cells (DCs), B-cells, CD4+ and CD8+ T-cells, and NK cells can be detected and quantified. This method gives detailed information about immune cells in AT and the amount of each specific subset. Since there are numerous fluorescent antibodies available, our flow cytometry approach can be adjusted to measure various other cellular and intracellular markers of interest.

This article describes a method to analyze immune cell content of adipose tissue by isolation of immune cells from adipose tissue and subsequent analysis using flow cytometry.

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the ...

JoVE Journal

Medicine

Segmentation of the Choroid Plexus Using Manual Delineations

To begin, import digital imaging and communications in medicine or DICOM data by clicking on the DCM button located at the upper left corner of the toolbar and clicking the import DICOM files button. To adjust the brightness of an image, click adjust window and level of volume or left click on the image and drag the mouse up or down. Then left click on the image slice and move the mouse to the left or right to adjust the contrast.Refer to the deep gray matter nuclei or the contrast scale bars signal intensity and ensure that it remains consistent throughout the segmentation to choose the suitable contrast for the choroid plexus. To start the segmentation of the lateral third and fourth ventricle choroid plexus, select the segment editor from the module's dropdown menu. Now click on the dropdown menu for segmentation to select different segmentations and rename the selected one.Use the master volume dropdown and pick the NIfTI or DICOM sets that need editing. Click the add button two times to include two segments for the lateral ventricle choroid plexus. Double click on their titles and rename them as right lateral ventricle choroid plexus and left lateral ventricle choroid plexus.Click on the add button once more to introduce segments for the choroid plexus of the third and fourth ventricles. Afterward, rename them as the third ventricle choroid plexus and the fourth ventricle choroid plexus respectively. Begin with the lateral ventricle choroid plexus in the axial plane and ensure to position images based on the bicommissural line.Use the trigonum collaterale as a reference point to locate the lateral ventricle choroid plexus. To start manual segmentation, select either the paint or draw tool located in the effects section of the segment editor. While using the paint tool, select the desired brush diameter for painting.Use the red nucleus level to determine when to stop segmenting the choroid plexus in the lateral ventricles. To locate the third ventricle choroid plexus, commence in the sagittal plane, and use the foramen of Monro, fornix, corpus callosum, thalamus, and internal cerebral vein as landmarks. Similarly, for the fourth ventricle choroid plexus, begin in the sagittal plane and use the superior cerebellar peduncle, pons and medulla as reference points to pinpoint the choroid plexus in the fourth ventricle.From the modules dropdown menu, navigate to quantification and select segment statistics. Then under the input section, select the newly created segmentation map for quantification under the segmentation tool and select the MRI volume from the scaler volume. For the output table, select the table option under output and hit apply.A table displaying the choroid plexus volume in different units will appear. Click on the save button located in the top left part of the toolbar to store the generated files. Preserve the segmentation files in the formats nrrd, nii.gz, or tsv.

Flow Cytometry and Data Analysis of Murine Brain Histone Post-Translational Modifications

Analyze the antibody panel, confirm that the cytometer has a minimum of four lasers, including red, yellow, blue, and violet. Establish the compensation matrix with either compensation beads or single stain cell controls. To calibrate and standardize, run rainbow fluorescent beads at the beginning of each experiment by adjusting the photomultiplier tube voltage until the bead peaks align with the target values from the previous experiments.Set the photomultiplier tube voltage and gain for the experiment. Then use antibody captured compensation beads to establish a compensation matrix. Next, plot the side scatter area on log versus the forward scatter height on linear in a dot plot, gate out debris and select for cell size using the S1 gate, choose singlet cells in a dot plot a forward scatter width versus forward scatter height with S2 gate.For establishing floor four gates, use the relevant FMO for each floor four channel. With single parameter histograms determine the positive signal in each channel and establish the gates accordingly. Carefully record the samples using the established gating strategy.Identify microglia using P2 RY 12 plus signal and determine protein expression in the respective channel for microglia only. Establish analysis gates on the cytometer analysis software user interface by replicating the same gating strategy used during recording. Use the add statistics function to select the median for the population of interest on the compensated channel height.Export the MFI values for the respective channels to a spreadsheet using the table editor for further statistical analysis. Lipopolysaccharide treatment induced an increase in histone three lysine 27 acetylation. When the MFI is normalized within sex, histogram analysis of the stained cells showed normally distributed populations with cell shifts to increased fluorescence.A similar increase in histone three lysine 27 acetylation was seen in the stained cells.

Isolating Immune Cells from Mouse Choroid Plexuses

Transcript