eoe sub articles

EoE Sub Articles

1. Flow cytometry measurement of MRC (mitochondrial respiratory chain) complex subunits and TFAM (mitochondrial transcription factor A) in fixed cells
<ol>
	<li>At the end of the culture period, detach the cells (~106) by adding the cell dissociation reagent; then, pellet and collect the cells in a 15 mL tube. Wash the cells by centrifugation with phosphate buffered saline (PBS) (1x) twice by centrifuging at 300 &#215; g for 5 min.</li>
	<li>Fix the cells in 1.6% paraformaldehyde (PFA) (1 mL of 1.6% PFA in a 15 mL tube) at room temperature (RT) for 10 min. Wash the cells by centrifugation with PBS (1x) twice by centrifuging at 300 &#215; g for 5 min.</li>
	<li>Permeabilize the cells with ice-cold 90% methanol (1 mL of 90% methanol in a 15 mL tube) at -20 &#176;C for 20 min.</li>
	<li>Block the samples in blocking buffer containing 0.3 M glycine, 5% goat serum, and 1% bovine serum albumin (BSA) - Fraction V in PBS (1x) (1 mL of blocking buffer in a 15 mL tube). Wash the cells by centrifugation with PBS (1x) twice.</li>
	<li>Incubate the cells with the following primary antibodies for 30 min: anti-NDUFB10 (1:1,000) for measurement of complex I subunit, anti-succinate dehydrogenase complex flavoprotein subunit A (SDHA, 1:1,000) for measurement of complex II subunit and anti-COX IV (1:1,000) for measurement of complex IV subunit, and anti-TFAM (mitochondrial transcription factor A) antibody conjugated with Alexa Fluor 488 (1:400). Stain the same number of cells separately with anti-TOMM20 (translocase of outer mitochondrial membrane 20) antibody conjugated with Alexa Fluor 488 (1:400) for 30 min (1 mL of primary antibody solution in a 15 mL tube; see the Table of Materials for details about the antibodies).</li>
	<li>Wash the cells with PBS (1x) once with centrifugation at 300 &#215; g for 5 min. Add secondary antibody (1:400) into tubes of NDUFB10, SDHA, and COX IV and incubate the cells with these solutions for 30 min.</li>
	<li>Wash the cells with PBS (1x) once by centrifuging at 300 &#215; g for 5 min. Aspirate the supernatants, leaving approximately 100 &#181;L in the tubes. Resuspend the cell pellets in 300 &#181;L of PBS (1x). Transfer the cells to 1.5 mL microcentrifuge tubes kept in the dark on ice.</li>
	<li>Analyze the cells on the flow cytometer (with a 3 blue and 1 red laser configuration). Detect signals in filter 1 (FL1) using a 530/30 bandpass filter.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>anti-TFAM conjugated with Alexa Fluor 488</td>
			<td>Abcam</td>
			<td>ab198308</td>
			<td>Primary Antibody; use as 1:400, 2.5 &#956;L in 1000 &#956;L staining solution; use mouse monoclonal IgG2b Alexa Fluor&#174; 488 as an isotype control.</td>
		</tr>
		<tr>
			<td>anti-TOMM20 conjugated with Alexa Fluor 488</td>
			<td>Santa Cruz Biotechnology</td>
			<td>Cat# sc-17764 RRID:AB_628381</td>
			<td>Primary Antibody; use as 1:400, 2.5 &#956;L in 1000 &#956;L staining solution; use mouse monoclonal IgG2a Alexa Fluor&#174; 488 as an isotype control.</td>
		</tr>
		<tr>
			<td>anti-NDUFB10</td>
			<td>Abcam</td>
			<td>ab196019</td>
			<td>Primary Antibody; use as 1:1000, 1 &#956;L in 1000 &#956;L staining solution; use Alexa Fluor &#174; 488 goat anti-rabbit IgG (1:400, Thermo Fisher Scientific, Catalog # A-11008) as secondary antibody; use rabbit monoclonal IgG as an isotype control.</td>
		</tr>
		<tr>
			<td>anti-SDHA</td>
			<td>Abcam</td>
			<td>ab137040</td>
			<td>Primary Antibody; use as 1:1000, 1 &#956;L in 1000 &#956;L staining solution; use Alexa Fluor &#174; 488 goat anti-rabbit IgG (1:400, Thermo Fisher Scientific, Catalog # A-11008) as secondary antibody; use rabbit monoclonal IgG as an isotype control.</td>
		</tr>
		<tr>
			<td>anti-COX IV</td>
			<td>Abcam</td>
			<td>ab14744, RRID:AB_301443</td>
			<td>Primary Antibody; use as 1:1000, 1 &#956;L in 1000 &#956;L staining solution; use Alexa Fluor &#174; 488 goat anti-mouse IgG (1:400, Thermo Fisher Scientific, Catalog # A-11001) as secondary antibody; use mouse monoclonal IgG as an isotype control.</td>
		</tr>
		<tr>
			<td>PBS 1x</td>
			<td>Thermo Fisher Scientific</td>
			<td>18912014</td>
			<td>Used for washing steps</td>
		</tr>
		<tr>
			<td>Formaldehyde (PFA) 16%</td>
			<td>Thermo Fisher Scientific</td>
			<td>28908</td>
			<td>Cell fixation</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>G8898</td>
			<td>Used in blocking buffer</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Europa Bioproducts</td>
			<td>EQBAH62-1000</td>
			<td>Blocking agent to prevent non-specific binding of antibodies in immunostaining assays and CDM ingredient</td>
		</tr>
	</tbody>
</table>

measurement of multiple mitochondrial parameters in neurons using flow cytometry

Source: Liang, K. X., et al. <a href="https://app.jove.com/v/63116">Flow Cytometric Analysis of Multiple Mitochondrial Parameters in Human Induced Pluripotent Stem Cells and Their Neural and Glial Derivatives.</a> J. Vis. Exp. (2021)
This video demonstrates the labeling of neuronal cells with antibodies for mitochondrial respiratory chain complexes, mitochondrial DNA-associated protein, and mitochondrial outer membrane protein, followed by flow cytometry to quantify their levels.

Measurement of Multiple Mitochondrial Parameters in Neurons using Flow Cytometry

Source: Liang, K. X., et al. Flow Cytometric Analysis of Multiple Mitochondrial Parameters in Human Induced Pluripotent Stem Cells and Their Neural and ...

Take fixed and permeabilized neurons in a tube.
Add a blocking buffer to prevent non-specific antibody binding.
Centrifuge the suspension and remove the supernatant. Then, resuspend the neurons in buffer.
Transfer the suspension equally into tubes containing untagged and fluorophore-tagged primary antibodies.
Incubate, allowing the antibodies to specifically bind to mitochondrial respiratory chain or MRC complex subunits, a mitochondrial DNA-associated protein, and a mitochondrial outer membrane protein within the neurons.
Remove any unbound antibodies.
Incubate with fluorophore-tagged secondary antibodies that bind to the untagged primary antibodies.
Centrifuge the mixture and discard the supernatant.
Resuspend the neurons in buffer and transfer them to microcentrifuge tubes.
Perform flow cytometry to detect the fluorescent signals on the labeled neurons, corresponding to the expression levels of MRC complex subunits and the measurement of relative mitochondrial DNA levels.

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32 Aufrufe. Source: Liang, K. X., et al. Flow Cytometric Analysis of Multiple Mitochondrial Parameters in Human Induced Pluripotent Stem Cells and Their Neural and Glial Derivatives. J. Vis. Exp. (2021) This video demonstrates the labeling of neuronal cells with antibodies for mitochondrial respiratory chain complexes, mitochondrial DNA-associated protein, and mitochondrial outer membrane protein, followed by flow cytometry to quantify their levels. 

Serie: Measurement of Multiple Mitochondrial Parameters in Neurons using Flow Cytometry - Konzept

Flow Cytometric Analysis of Mitochondrial Reactive Oxygen Species in Murine Hematopoietic Stem and Progenitor Cells and MLL-AF9 Driven Leukemia

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from healthy mice as well as mice with AML driven by MLL-AF9. Specifically, we describe a two-step cell staining process, whereby healthy or leukemia BM cells are first stained with a fluorogenic dye that detects mitochondrial superoxides, followed by staining with fluorochrome-linked monoclonal antibodies that are used to distinguish various healthy and malignant hematopoietic progenitor populations. We also provide a strategy for acquiring and analyzing the samples by flow cytometry. The entire protocol can be carried out in a timeframe as short as 3-4 h. We also highlight the key variables to consider as well as the advantages and limitations of monitoring ROS production in the mitochondrial compartment of live hematopoietic and leukemia stem and progenitor subpopulations using fluorogenic dyes by flow cytometry. Furthermore, we present data that mitochondrial ROS abundance varies among distinct healthy HSPC sub-populations and leukemia progenitors and discuss the possible applications of this technique in hematologic research.

We describe a method for using multiparameter flow cytometry to detect mitochondrial reactive oxygen species (ROS) in murine healthy hematopoietic stem and progenitor cells (HSPCs) and leukemia cells from a mouse model of acute myeloid leukemia (AML) driven by MLL-AF9.

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from ...

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Flow Cytometric Analysis of Apoptotic Biomarkers in Actinomycin D-Treated SiHa Cervical Cancer Cells

Apoptosis biomarkers were investigated in actinomycin D-treated SiHa cervical cancer cells using a benchtop flow cytometer. Early biomarkers (Annexin V and mitochondrial membrane potential) and late biomarkers (caspases 3 and 7, and DNA damage) of apoptosis were measured in experimental and control cultures. Cultures were incubated for 24 hours in a humidified incubator at 37 &#176;C with 5% CO2. The cells were then detached using trypsin and enumerated using a flow cytometric cell count assay. Cells were further analyzed for apoptosis using an Annexin V assay, a mitochondrial electrochemical transmembrane potential assay, a caspase 3/7 assay, and a DNA damage assay. This article provides an overview of apoptosis and traditional flow cytometry, and elaborates flow cytometric protocols for processing and analyzing SiHa cells. The results describe positive, negative, and sub-optimal experimental data. Also discussed are interpretation and caveats in performing flow cytometric analysis of apoptosis using this analytical platform. Flow cytometric analysis provides an accurate measurement of early and late biomarkers for apoptosis.

Apoptosis can be characterized by flow cytometric analysis of early and late apoptotic biomarkers. The cervical cancer cell line, SiHa, was analyzed for apoptosis biomarkers after treatment with Actinomycin D using a benchtop flow cytometer.

Durchflusszytometrische Analyse von apoptotischen Biomarkern in Actinomycin-D-behandelten SiHa-Gebärmutterhalskrebszellen

Apoptosis biomarkers were investigated in actinomycin D-treated SiHa cervical cancer cells using a benchtop flow cytometer. Early biomarkers (Annexin V ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Isolation und Analyse über Durchflusszytometrie von Immunzellen aus dem ischämischen Gehirn der Maus

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Measurement of Multiple Mitochondrial Parameters in Neurons using Flow Cytometry

Transkript

Measurement of Multiple Mitochondrial Parameters in Neurons using Flow Cytometry

Flow Cytometric Analysis of Mitochondrial Reactive Oxygen Species in Murine Hematopoietic Stem and Progenitor Cells and MLL-AF9 Driven Leukemia

Durchflusszytometrische Analyse von apoptotischen Biomarkern in Actinomycin-D-behandelten SiHa-Gebärmutterhalskrebszellen

Isolation und Analyse über Durchflusszytometrie von Immunzellen aus dem ischämischen Gehirn der Maus