eoe sub articles

EoE Sub Articles

1. Flow cytometry staining of Mtb-infected monocyte-derived cells
NOTE: The following steps must be performed in a BSL-3 facility. The flow cytometry staining could be performed in a 96-well plate instead of tubes.
<ol>
	<li>Detach the Mycobacterium tuberculosis, Mtb-infected cells (and uninfected controls) from the wells in the 6 well plate(s) by incubation with 1 mL of FACS buffer per well for at least 30 min at 37 &#176;C and 5% CO2.</li>
	<li>Gently pipette up and down a few times to ensure that the cells are detached. If possible, confirm cell detachment with microscopy. Transfer the cell suspension from each well to a screw-capped microcentrifuge tube and spin the tubes at 200 x g for 5 min. Discard the supernatant carefully by pipetting.</li>
	<li>Wash the cell pellet in each tube twice with FACS buffer and spin the cells at 200 x g for 5 min.</li>
	<li>Stain the cells (about 0.5 x 106 to 1 x 106 cells/tube) with approximately 50 &#181;L cocktail of fluorochrome-conjugated anti-human antibodies including TLR2 (AF647), CD206 (APC-Cy7), CD163 (BV605), CD80 (BV650), CCR7 (BV711), CD86 (BV786), CD200R (PE), CD64 (PE-Dazzle 594), HLA-DR (PE-Cy5) (Table 1) in combination with viability dye Zombie-UV for 30 min at 4 &#176;C (refrigerator) in the dark.</li>
	<li>Wash the stained cells twice with 400 &#181;L of FACS buffer and spin the cells at 200 x g for 5 min.</li>
	<li>Fix the stained cells with 200 &#181;L of fixation buffer (freshly prepared) for 30 min at RT in the dark to ensure complete inactivation of mycobacteria.</li>
	<li>Wash the cells twice with 400 &#181;L of FACS buffer and spin at 200 x g for 5 min to remove excess fix buffer.</li>
	<li>Resuspend the fixed cells in 400 &#181;L of FACS buffer and transfer the samples into new 1 mL microcentrifuge tubes before taking them out of the BSL-3 laboratory for flow cytometry in BSL-2. Store the stained cells at +4 &#730;C until sample acquisition. 
	NOTE: Spray the tubes with 70% ethanol before taking them out of the BSL-3 laboratory. Formaldehyde is toxic (carcinogenic) and must be handled in a class II biosafety cabinet. Discard formaldehyde waste in a separate chemical waste.</li>
</ol>
2. Flow cytometric data acquisition and analysis of Mtb-infected monocyte-derived cells
NOTE: Steps 2.1&#8211;2.2 should be performed in advance of the flow cytometry staining described above. To avoid problems with cell clumping and dissociation of tandem dyes after cell fixation, sample acquisition of both Mtb-infected and uninfected cells is performed within 4-10 h after primary antibody staining.
<ol>
	<li>Before flow cytometry staining described above, compensate the fluorescent signal for each fluorochrome-conjugated antibody listed in the staining panel (Table 1) using compensation beads (both positive and negative).</li>
	<li>Titrate the antibody dilution for staining of human macrophages to obtain the optimal signal for each fluorochrome.</li>
	<li>Use unstained cells to determine the level of background fluorescence necessary to set a gate for the negative cell population allowing for the stained cells to be visualized (macrophages are highly auto-fluorescent).</li>
	<li>Acquire a minimum of 50,000 cells/sample in the flow cytometer using the recommended software for data acquisition.</li>
	<li>Export the acquisition files from the flow cytometer in flow cytometry standard (FCS) format 3.1.</li>
	<li>Analyze the FCS files in flow cytometry analysis software.</li>
	<li>Gate macrophages according to their forward- and side scatter (FSC and SSC) characteristics and exclude dead cells by live/dead cell gating using the Zombie-UV viability dye.</li>
	<li>Visualize H37Rv-GFP infected macrophages in the FITC channel.</li>
	<li>Identify the frequency of positively stained cells and geometric mean fluorescence intensity (MFI) for all markers (Table 1).</li>
</ol>
Table 1: List of antibodies used for flow cytometry.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Laser</td>
			<td>Filter</td>
			<td>Fluorochrome</td>
			<td>Phenotype</td>
			<td>Function</td>
			<td>Clone</td>
			<td>Catalog no.</td>
			<td>Company</td>
		</tr>
		<tr>
			<td>639</td>
			<td>670/30</td>
			<td>AF647</td>
			<td>TLR2</td>
			<td>Pathogen recognition receptor</td>
			<td>TL2.1</td>
			<td>309714</td>
			<td>BioLegend</td>
		</tr>
		<tr>
			<td>639</td>
			<td>780/60</td>
			<td>APC-Cy7</td>
			<td>CD206</td>
			<td>Mannose receptor</td>
			<td>15-2</td>
			<td>321120</td>
			<td>BioLegend</td>
		</tr>
		<tr>
			<td>405</td>
			<td>610/20</td>
			<td>BV605</td>
			<td>CD163</td>
			<td>Scavenger receptor</td>
			<td>GHI/61</td>
			<td>333616</td>
			<td>BioLegend</td>
		</tr>
		<tr>
			<td>405</td>
			<td>670/30</td>
			<td>BV650</td>
			<td>CD80</td>
			<td>Co-stimulatory molecule</td>
			<td>2D10</td>
			<td>305227</td>
			<td>BioLegend</td>
		</tr>
		<tr>
			<td>405</td>
			<td>710/50</td>
			<td>BV711</td>
			<td>CCR7</td>
			<td>Chemokine receptor</td>
			<td>G043H7</td>
			<td>353228</td>
			<td>BioLegend</td>
		</tr>
		<tr>
			<td>405</td>
			<td>780/60</td>
			<td>BV785</td>
			<td>CD86</td>
			<td>Co-stimulatory molecule</td>
			<td>IT2.2</td>
			<td>305442</td>
			<td>BioLegend</td>
		</tr>
		<tr>
			<td>488</td>
			<td>530/30</td>
			<td>GFP</td>
			<td>Mtb</td>
			<td>Intracellular bacteria</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>561</td>
			<td>586/15</td>
			<td>PE</td>
			<td>CD200R</td>
			<td>Inhibitory receptor</td>
			<td>OX-108</td>
			<td>329306</td>
			<td>BioLegend</td>
		</tr>
		<tr>
			<td>561</td>
			<td>620/14</td>
			<td>PE/DAZZLE 594</td>
			<td>CD64</td>
			<td>Fc gamma receptor-I of IgG</td>
			<td>10.1</td>
			<td>305032</td>
			<td>BioLegend</td>
		</tr>
		<tr>
			<td>561</td>
			<td>661/20</td>
			<td>PE-Cy5 (PC5)</td>
			<td>HLA-DR</td>
			<td>MHC class II molecule</td>
			<td>L243</td>
			<td>307608</td>
			<td>BioLegend</td>
		</tr>
		<tr>
			<td>355</td>
			<td>450/50</td>
			<td>BUV395</td>
			<td>Viability Dye</td>
			<td>Live/dead cell marker</td>
			<td>Zombie UV</td>
			<td>423108</td>
			<td>Invitrogen</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Formaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>F8775</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG Alexa Fluor 594 secondary antibody</td>
			<td>Invitrogen</td>
			<td>R37121</td>
			<td>Secondary antibody for CD64</td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG Alexa Fluor 594 secondary antibody</td>
			<td></td>
			<td>A-11037</td>
			<td>Secondary antibody for CD163</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Sigma-Aldrich</td>
			<td>F7524</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA (0.5 M)</td>
			<td>Karolinska University hospital, Huddinge</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Comp bead plus</td>
			<td>BD</td>
			<td>560497</td>
			<td></td>
		</tr>
	</tbody>
</table>

flow cytometry staining of mycobacterium tuberculosis infected macrophage subpopulations

Source: Mily, A., et al. <a href="https://app.jove.com/v/61807">Polarization of M1 and M2 Human Monocyte-Derived Cells and Analysis with Flow Cytometry upon Mycobacterium tuberculosis Infection.</a> J. Vis. Exp. (2020)
This video showcases a flow cytometric gating strategy utilizing a multi-color panel to analyze cell viability and specific fluorescent-conjugated anti-human antibody markers. This approach makes it possible to determine the percentage of Mycobacterium tuberculosis-infected cell populations accurately.

Flow Cytometry Staining of Mycobacterium tuberculosis Infected Macrophage Subpopulations

1. Flow cytometry staining of Mtb-infected monocyte-derived cells
NOTE: The following steps must be performed in a BSL-3 facility. The flow cytometry ...

Begin with adherent M1 and M2 macrophage subpopulations, distinguished by their specific surface markers. These cells are infected with fluorescent-labeled Mycobacterium tuberculosis.
Add FACS buffer to the culture wells.
EDTA in the buffer enables cell detachment from the culture wells.
Transfer the cell suspension to microcentrifuge tubes.
Centrifuge the tubes. Discard the supernatant. Resuspend the cells in a fluorochrome-conjugated anti-macrophage antibody cocktail and viability dye, Zombie-UV.
The Zombie-UV fluorescence dye enters dead cells through damaged membranes and binds to cytoplasmic proteins, enabling clear differentiation from viable unstained cells.
Further, the fluorescent-conjugated anti-macrophage antibodies bind to target antigens on the macrophages, enabling differential detection of subpopulations.
Wash the cells to remove unbound antibodies. Centrifuge. Remove the supernatant.
Resuspend the cells in a fixative buffer to cross-link proteins, preserving cell morphology, and inactivating Mycobacteria for safe handling during analysis.
Finally, centrifuge and process the samples for flow cytometry.
Use an appropriate gating strategy to determine the percentage of Mycobacterium-infected subpopulations.

flow-cytometry-staining-mycobacterium-tuberculosis-infected

Research

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Cellular Immunodiagnostics

215 次观看. 来源:Mily， A. 等人 M1 和 M2 人单核细胞衍生细胞的极化以及流式细胞术对结核分枝杆菌感染的分析。J. Vis. Exp. （2020 年） 该视频展示了一种流式细胞术设门策略，该策略利用多色面板来分析细胞活力和特异性荧光偶联的抗人抗体标志物。这种方法可以准确测定结核分枝杆菌感染细胞群的百分比。 

视频: 结核分枝杆菌感染的巨噬细胞亚群的流式细胞术染色 - 概念

Isolation and Identification of Extravascular Immune Cells of the Heart

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with functional compartmentalization both during steady state and during different forms of inflammation. Until recently, the study of immune cells in the heart required the use of microscopy or poorly developed digestion protocols, which provided enough sensitivity during severe inflammation but were unable to confidently identify small &#8212; but key &#8212; populations of cells during steady state. Here, we discuss a simple method combining enzymatic (collagenase, hyaluronidase and DNAse) and mechanical digestion of murine hearts preceded by intravascular administration of fluorescently-labelled antibodies to differentiate small but unavoidable intravascular cell contaminants. This method generates a suspension of isolated viable cells that can be analyzed by flow cytometry for identification, phenotyping and quantification, or further purified with fluorescence-activated cell sorting or magnetic bead separation for transcriptional analysis or in vitro studies. We include an example of a step-by-step flow cytometric analysis to differentiate the key macrophage and dendritic cell populations of the heart. For a medium sized experiment (10 hearts) the completion of the procedure requires 2&#8211;3 h.

This protocol presents a simple and efficient method to isolate, identify and quantify immune cells residing in the myocardium of mice during steady state or inflammation. The protocol combines enzymatic and mechanical digestion for the generation of a single cell suspension that can be further analyzed by flow cytometry.

心脏外免疫细胞的分离与鉴定

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with ...

科研

JoVE Journal

免疫与感染

Flow Cytometry-Based Quantification and Analysis of Myocardial B-Cells

A growing body of evidence shows that B-lymphocytes play an important role in the context of myocardial physiology and myocardial adaptation to injury. However, the literature reports contrasting data on the prevalence of myocardial B-cells. B-cells have been reported to be both among the most prevalent immune cells in the rodent heart or to be present, but at a markedly lower prevalence than myeloid cells, or to be quite rare. Similarly, several groups have described that the number of myocardial B-cells increases after acute ischemic myocardial injury, but one group reported no changes in the number of B-cells of the injured myocardium. Implementation of a shared, reproducible method to assess the prevalence of myocardial B-cells is critical to harmonize observations from different research groups and thus promote the advancement of the study of B-cell myocardial interactions. Based on our experience, the seemingly contrasting observations reported in the literature likely stem from the fact that murine myocardial B-cells are mostly intravascular and connected to the microvascular endothelium. Therefore, the number of B-cells recovered from a murine heart is exquisitely sensitive to the perfusion conditions used to clean the organ and to the method of digestion used. Here we report an optimized protocol that accounts for these two critical variables in a specific way. This protocol empowers reproducible, flow cytometry-based analysis of the number of murine myocardial B-cells and allows researchers to distinguish extravascular vs. intravascular myocardial B-cells.

Here we report a protocol for the quantification and differentiation of myocardial B-lymphocytes based on their location in the intravascular or endothelial space using flow cytometry.

基于流式细胞术的心肌B细胞定量和分析

A growing body of evidence shows that B-lymphocytes play an important role in the context of myocardial physiology and myocardial adaptation to injury. ...

Flow Cytometric Analysis of Extracellular Vesicles from Cell-conditioned Media

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for phenotypic analyses of extracellular vesicles (EV) including exosomes (Exo) in the peripheral blood and other body fluids. The small size of EV mandates the use of dedicated instruments having a detection threshold around 50-100 nm. Alternatively, EV can be bound to latex microbeads that can be detected by FC. Microbeads, conjugated with antibodies that recognize EV-associated markers/Cluster of Differentiation CD63, CD9, and CD81 can be used for EV capture. Exo isolated from CM can be analyzed with or without pre-enrichment by ultracentrifugation. This approach is suitable for EV analyses using conventional FC instruments. Our results demonstrate a linear correlation between Mean Fluorescence Intensity (MFI) values and EV concentration. Disrupting EV through sonication dramatically decreased MFI, indicating that the method does not detect membrane debris. We report an accurate and reliable method for the analysis of EV surface antigens, which can be easily implemented in any laboratory.

The protocol describes a reproducible method designed for use with cell culture supernatants to detect surface epitopes on small extracellular vesicles (EV). It utilizes specific EV immunoprecipitation using beads coupled with antibodies that recognize surface antigen CD9, CD63, and CD81. The method is optimized for downstream flow cytometry analysis.

细胞调理介质细胞外囊泡的流式细胞学分析

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for ...

Flow Cytometry Staining of Mycobacterium tuberculosis Infected Macrophage Subpopulations

成績單

Flow Cytometry Staining of Mycobacterium tuberculosis Infected Macrophage Subpopulations