eoe sub articles

EoE Sub Articles

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1-200 &#956;L universal fit bulk packed pipet tips</td>
			<td>Corning</td>
			<td>CLS4866-1000EA</td>
			<td></td>
		</tr>
		<tr>
			<td>2.4G2 anti-Fc&#947;R mAb</td>
			<td>BD</td>
			<td>553141</td>
			<td>10 &#956;g/ml final concentration</td>
		</tr>
		<tr>
			<td>5 ml syringe plunger</td>
			<td>BD Emerald</td>
			<td>307733</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml conical tubes</td>
			<td>MercK Millipore</td>
			<td>SBHA025SB</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm TC-treated Cell Culture Dish</td>
			<td>Falcon</td>
			<td>353002</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#956;m cell strainer</td>
			<td>Falcon</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well Clear Round Bottom TC-treated Culture Microplate</td>
			<td>Falcon</td>
			<td>353077</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Rat/Hamster Ig,k/Negative Control Compensation Particles</td>
			<td>BD- Bioscience</td>
			<td>552845</td>
			<td></td>
		</tr>
		<tr>
			<td>Beta-mercaptoethanol</td>
			<td>Sigma</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A07030</td>
			<td></td>
		</tr>
		<tr>
			<td>BUV805 Rat Anti-Mouse CD8a</td>
			<td>BD- Bioscience</td>
			<td>564920</td>
			<td>4 &#956;g/ml final concentration</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffer Saline w/o Calcium w/o Magnesium</td>
			<td>Euroclone</td>
			<td>ECB4004L</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Safe-Lock Tubes, 1.5 mL</td>
			<td>Eppendorf</td>
			<td>30120159</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma</td>
			<td>34852-1L-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic Acid Disodium Salt solution (EDTA)</td>
			<td>Sigma</td>
			<td>E7889</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Corning</td>
			<td>35-079-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Filcon, Sterile, Syringe-Type 70 &#956;m</td>
			<td>Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixable Viability Dye eFluor 780</td>
			<td>eBioscience</td>
			<td>65-0865-14</td>
			<td>1:1000 final concentration</td>
		</tr>
		<tr>
			<td>Foxp3 / Transcription Factor Staining Buffer Set</td>
			<td>eBioscience</td>
			<td>00-5523-00</td>
			<td>This Set contains fixation/permeabilization concentrate and diluent, and permeabilization buffer 10x</td>
		</tr>
		<tr>
			<td>H-2k(d) AMQMLKETI allophycocyanin (APC)-labelled tetramer</td>
			<td>provided by NIH Tetramer Core Facility</td>
			<td></td>
			<td>6 &#956;g/ml final concentration</td>
		</tr>
		<tr>
			<td>H-2k(d) AMQMLKETI phycoerythrine (PE) labelled pentamer</td>
			<td>Proimmune</td>
			<td>F176-2A-E - 176</td>
			<td>10 &#956;L / sample</td>
		</tr>
		<tr>
			<td>Hoechst 33342, Trihydrochloride, Trihydrate - 10 mg/mL Solution in Water</td>
			<td>ThermoFisher</td>
			<td>H3570</td>
			<td></td>
		</tr>
		<tr>
			<td>Ki-67 Monoclonal Antibody (SolA15), FITC</td>
			<td>eBioscience</td>
			<td>11-5698-82</td>
			<td>5 &#956;g/ml final concentration</td>
		</tr>
		<tr>
			<td>L-Glutamine 100X (200 mM)</td>
			<td>Euroclone</td>
			<td>ECB3000D</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex-HA Filters 0,45 &#181;m</td>
			<td>BD</td>
			<td>340606</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin 100X</td>
			<td>Euroclone</td>
			<td>ECB3001D</td>
			<td></td>
		</tr>
		<tr>
			<td>PE/Cyanine7 anti-mouse CD62L Antibody</td>
			<td>Biolegend</td>
			<td>104418</td>
			<td>0.2 &#956;g/ml final concentration</td>
		</tr>
		<tr>
			<td>PerCP-Cy&#8482;5.5 Hamster Anti-Mouse CD3e</td>
			<td>BD- Bioscience</td>
			<td>551163</td>
			<td>4.4 &#956;g/ml final concentration</td>
		</tr>
		<tr>
			<td>Red Blood Cell Lysis Buffer</td>
			<td>Sigma</td>
			<td>R7757</td>
			<td></td>
		</tr>
		<tr>
			<td>Round-Bottom Polystyrene Tubes, 5 mL</td>
			<td>Falcon</td>
			<td>352058</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium without L-Glutamine with Phenol Red</td>
			<td>Euroclone</td>
			<td>ECB9006L</td>
			<td></td>
		</tr>
		<tr>
			<td>Software package for analyzing flow cytometry data</td>
			<td>FlowJo</td>
			<td>v.10</td>
			<td></td>
		</tr>
		<tr>
			<td>Software for acquisition of samples at flowcytometer</td>
			<td>BD FACSDiva</td>
			<td>v 6.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Solution</td>
			<td>Euroclone</td>
			<td>ECM0990D</td>
			<td></td>
		</tr>
	</tbody>
</table>

an assay for cell cycle analysis of antigen-specific cd8+ t cells

Source: Simonetti, S., et al. <a href="https://app.jove.com/v/61867">A DNA/Ki67-Based Flow Cytometry Assay for Cell Cycle Analysis of Antigen-Specific CD8 T Cells in Vaccinated Mice.</a> J. Vis. Exp. (2021)
This video demonstrates an assay to analyze antigen-specific CD8+ T cells in different cell cycle stages. Fluorescently-labeled viable cells are selected using flow cytometry, and the cells in the different cell cycle stages are identified by their DNA content using antibodies against cell proliferation-associated nuclear protein and a nucleic acid stain.

<img alt="Figure 1" src="/files/ftp_upload/21985/21985_Figure1.jpg" /> 
Figure 1: Cell cycle analysis of BM cells. BM cells from untreated Balb/c mice were stained and analyzed by flow cytometry. (A) Example of gating strategy. We gated on single cells in the DNA-A/-W plot (left) and subsequently on live cells by dead cell dye exclusion (middle). Then, a &#34;relaxed&#34; FSC-A/SSC-A gate was used for all BM cells (right). (B) Example of cell cycle analysis of BM cel...

An Assay for Cell Cycle Analysis of Antigen-Specific CD8+ T Cells

1. Preparation of medium and staining solution

	Prepare Complete Medium: Roswell Park Memorial Institute (RPMI) medium with 2 mM glutamine, 100 U/mL ...

Take secondary lymphoid organ cells from an antigen-immunized mouse, containing antigen-specific CD8+ T cells in different cell cycle stages.
G1, S, G2, and M are stages of actively dividing cells, while cells in the G0 stage are quiescent.
Add a fluorescent viability dye to label dead cells, and fluorophore-tagged antibodies binding to T cell surface markers CD3 and CD8.
Introduce fluorophore-conjugated antigenic peptide-MHC multimers to bind T cell receptors, labeling the antigen-specific T cells.
Using a buffer, fix and permeabilize the cells.
Introduce fluorophore-tagged antibodies to bind a cell proliferation-associated nuclear protein &#8212; staining the actively dividing cells.
Add a fluorescent stain to label DNA.
Using flow cytometry, select the living cells and identify the antigen-specific CD8+ T cells via the surface-bound labels.
The DNA stain intensity discriminates G0 and G1 cells with a lower DNA amount from the S, G2, and M cells with a higher DNA amount. The absence of the nuclear protein-specific antibody separates the G0 cells.

an-assay-for-cell-cycle-analysis-of-antigen-specific-cd8-t-cells

Research

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Cellular Immunodiagnostics

86 Views. Source: Simonetti, S., et al. A DNA/Ki67-Based Flow Cytometry Assay for Cell Cycle Analysis of Antigen-Specific CD8 T Cells in Vaccinated Mice. J. Vis. Exp. (2021) This video demonstrates an assay to analyze antigen-specific CD8+ T cells in different cell cycle stages. Fluorescently-labeled viable cells are selected using flow cytometry, and the cells in the different cell cycle stages are identified by their DNA content using antibodies against cell proliferation-associated nuclear protein and...

Video: An Assay for Cell Cycle Analysis of Antigen-Specific CD8+ T Cells - Concept

Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and cytokinesis) and acytokinetic mitosis (karyokinesis but no cytokinesis). Such atypical cell cycle variations result in polyploid and multinucleated cells rather than in cell division. Therefore, to determine cardiac turnover and regeneration, it is of crucial importance to correctly identify cardiomyocyte nuclei, the number of nuclei per cell, and their cell cycle status. This is especially true for the use of nuclear markers for identifying cell cycle activity, such as thymidine analogues Ki-67, PCNA, or pHH3. Here, we present methods for recognizing cardiomyocytes and their nuclearity and for determining their cell cycle activity. We use two published transgenic systems: the Myh6-H2B-mCh transgenic mouse line, for the unequivocal identification of cardiomyocyte nuclei, and the CAG-eGFP-anillin mouse line, for distinguishing cell division from cell cycle variations. Combined together, these two systems ease the study of cardiac regeneration and plasticity.

To distinguish cell division from cell cycle variations in cardiomyocytes, we present protocols using two transgenic mouse lines: Myh6-H2B-mCh transgenic mice, for the unequivocal identification of cardiomyocyte nuclei, and CAG-eGFP-anillin mice, for distinguishing cell division from cell cycle variations.

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and ...

JoVE Journal

Developmental Biology

Cell Cycle-specific Measurement of &#947;H2AX and Apoptosis After Genotoxic Stress by Flow Cytometry

The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer treatments as used in clinical oncology. It enables a quantitative and longitudinal analysis of the DNA damage response after genotoxic stress, as induced by radiotherapy and a multitude of anti-cancer drugs. The method covers all stages of the DNA damage response, providing endpoints for induction and repair of DNA double-strand breaks (DSBs), cell cycle arrest and cell death by apoptosis in case of repair failure. Combining these measurements provides information about cell cycle-dependent treatment effects and thus allows an in-depth study of the interplay between cellular proliferation and coping mechanisms against DNA damage. As the effect of many cancer therapeutics including chemotherapeutic agents and ionizing radiation is limited to or strongly varies according to specific cell cycle phases, correlative analyses rely on a robust and feasible method to assess the treatment effects on the DNA in a cell cycle-specific manner. This is not possible with single-endpoint assays and an important advantage of the presented method. The method is not restricted to any particular cell line and has been thoroughly tested in a multitude of tumor and normal tissue cell lines. It can be widely applied as a comprehensive genotoxicity assay in many fields of oncology besides radio-oncology, including environmental risk factor assessment, drug screening and evaluation of genetic instability in tumor cells.

The presented method combines the quantitative analysis of DNA double-strand breaks (DSBs), cell cycle distribution and apoptosis to enable cell cycle-specific evaluation of DSB induction and repair as well as the consequences of repair failure.

The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer ...

Cancer Research

Use of the Pyrimidine Analog, 5-Iodo-2&prime;-Deoxyuridine (IdU) with Cell Cycle Markers to Establish Cell Cycle Phases in a Mass Cytometry Platform

The regulation of cell cycle phase is an important aspect of cellular proliferation and homeostasis. Disruption of the regulatory mechanisms governing the cell cycle is a feature of a number of diseases, including cancer. Study of the cell cycle necessitates the ability to define the number of cells in each portion of cell cycle progression as well as to clearly delineate between each cell cycle phase. The advent of mass cytometry (MCM) provides tremendous potential for high throughput single cell analysis through direct measurements of elemental isotopes, and the development of a method to measure the cell cycle state by MCM further extends the utility of MCM. Here we describe a method that directly measures 5-iodo-2&#8242;-deoxyuridine (IdU), similar to 5-bromo-2&#180;-deoxyuridine (BrdU), in an MCM system. Use of this IdU-based MCM provides several advantages. First, IdU is rapidly incorporated into DNA during its synthesis, allowing reliable measurement of cells in the S-phase with incubations as short as 10-15 minutes. Second, IdU is measured without the need for secondary antibodies or the need for DNA degradation. Third, IdU staining can be easily combined with measurement of cyclin B1, phosphorylated retinoblastoma protein (pRb), and phosphorylated histone H3 (pHH3), which collectively provides clear delineation of the five cell cycle phases. Combination of these cell cycle markers with the high number of parameters possible with MCM allow combination with numerous other metrics.

Use of the Pyrimidine Analog, 5-Iodo-2&#8242;-Deoxyuridine IdU with Cell Cycle Markers to Establish Cell Cycle Phases in a Mass Cytometry Platform

This protocol adapts cell cycle measurements for use in a mass cytometry platform. With the multi-parameter capabilities of mass cytometry, direct measurement of iodine incorporation allows identification of cells in S-phase while intracellular cycling markers enable characterization of each cell cycle state in a range of experimental conditions.

The regulation of cell cycle phase is an important aspect of cellular proliferation and homeostasis. Disruption of the regulatory mechanisms governing the ...

An Assay for Cell Cycle Analysis of Antigen-Specific CD8⁺ T Cells

Transcript

An Assay for Cell Cycle Analysis of Antigen-Specific CD8⁺ T Cells

Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes

Cell Cycle-specific Measurement of γH2AX and Apoptosis After Genotoxic Stress by Flow Cytometry

Use of the Pyrimidine Analog, 5-Iodo-2′-Deoxyuridine (IdU) with Cell Cycle Markers to Establish Cell Cycle Phases in a Mass Cytometry Platform