eoe sub articles

EoE Sub Articles

1. Barcode of Lysed/Fixed Blood Cells
<ol>
	<li>Thaw the lysed/fixed cell samples from the -80 &#176;C storage slowly on ice; a maximum of 20 samples can be labeled with unique barcodes and pooled using this system. Dilute the 10X barcoding perm buffer by 1:10 with PBS; make enough buffer for ~3 mL per sample.</li>
	<li>Fill one non-sterile trough with the CSM and one with the 1X barcoding perm buffer. Add 1 mL of ice-cold CSM to freshly thawed samples, mix thoroughly with a pipette, and transfer to the respective pre-labeled polypropylene cluster tubes.</li>
	<li>Take a 10 &#956;L sample to count cells using an automated cell counter or hemocytometer. Normalize the cell counts to 1.5&#8211;2 x 106 cells per sample in each cluster tube: remove and discard the volume of excess cells above 2 x 106 cells.</li>
	<li>Centrifuge the cells at 600 x g for 5 min at room temperature. Resuspend the cells in 1 mL of 1X barcoding perm buffer with a multichannel pipette and centrifuge at 600 x g for 5 min at room temperature. Aspirate off the supernatant.</li>
	<li>Line up the cluster tubes on a rack in the same order as indicated on the barcode key so the sample matches with its barcode. Add 800 &#956;L 1X barcoding perm buffer by multichannel pipette to all samples in the cluster tubes without touching the cell pellet with the pipette tips (no mixing) to reduce cell loss. Set aside the rack with the cluster tubes.</li>
	<li>Remove 20-plex Pd Barcoding Kit tube strips from -20 &#176;C and thaw at room temperature. Add 100 &#956;L of 1X barcoding perm buffer, mix thoroughly, and transfer 120 &#956;L of the resuspended barcode mix into the corresponding cell samples in the cluster tubes.</li>
	<li>Mix thoroughly by multichannel pipette so that there is no cross-contamination between individually barcoded samples. Incubate cluster tubes for 30 min at room temperature to allow the barcodes to label the cells.</li>
	<li>Centrifuge at 600 x g for 5 min at room temperature. Aspirate the supernatant, then resuspend in 1 mL of CSM.</li>
	<li>Centrifuge and resuspend in CSM again. 
	Note: Each sample is now labeled with a unique barcode, and samples are ready to be pooled.</li>
	<li>Centrifuge at 600 x g for 5 min at room temperature and aspirate the supernatant. With a single pipette and using the same tip, transfer all cell pellets in ~70&#8211;80 &#956;L residual volume to one polystyrene tube. Do NOT eject the pipette tip; set aside the single pipette with this tip.</li>
	<li>With a multichannel pipette and new tips, add ~100 &#956;L CSM to each original cluster tube to maximize cell recovery. With the single pipette with the tip that was set aside, transfer all cell pellets in ~100 &#956;L residual volume to the same polystyrene tube.</li>
	<li>Add CSM to top off the polystyrene tube (~3 mL). Count and record the cell number of the pooled barcode set. Centrifuge at 600 x g for 5 min at room temperature and aspirate the supernatant. Proceed to stain the barcoded samples on the same day. 
	Note: It is normal to expect ~20&#8211;30% cell loss in the barcoding process.</li>
</ol>
2. Staining of Barcoded Lysed/Fixed Blood Cells and Preparation for Analysis on Mass Cytometry Instrument
Note: Each 1X titer of staining antibody (1 &#956;L of antibody per 100 &#956;L staining reaction), can usually stain 3&#8211;4 x 106 cells. Therefore, when all barcoded samples are pooled into one tube, the amount of antibody must be scaled up. If 20 barcoded samples amount to 30 x 106 cells, and each 1X titer can stain 3&#8211;4 x 106 cells, the barcoded sample only requires a 10X titer, as opposed to staining each sample individually, which would require a 20X amount of antibody (1X per individual tube). The concentration of the antibody to cell number should be carefully titrated for each individual antibody cocktail (not discussed here).
<ol>
	<li>Stain the cells using antibodies (Table of Materials) for surface staining and cytokine induction.
	<ol>
		<li>Make the surface staining cocktail by calculating the amount to be added and accounting for pipetting error (i.e., if staining 20 barcoded samples of 2 x 106 cells in each sample, a 10X titer stain is required; for final volume calculations, compensate for pipetting error by making a 10.5X final staining solution).</li>
		<li>Add 10X worth of surface staining volume to the barcoded cell pellet. To reduce cell loss, with the same tip, mix and measure up the volume of surface stains and cell pellets.</li>
		<li>Based on the volume of cells and staining cocktail, add CSM to a final staining volume of 50 &#956;L per number of cell samples (i.e., if running a set of 20 samples, 50 &#956;L X 20 = 1 mL). Incubate for 30 min at 4 &#176;C. Agitate the sample every 15 min to promote even staining.</li>
		<li>During the surface stain incubation, prepare the Perm/Wash buffer (Table of Materials) by diluting 1:10 with filtered ddH2O. Prepare volumes of 2 mL for permeabilization and 5 mL for washing. Keep at 4 &#176;C or on ice.</li>
		<li>Top off the surface staining tube with CSM, and centrifuge at 600 x g at 4 &#176;C for 5 min. Aspirate the supernatant. Resuspend the barcoded sample in 2 mL of 4 &#176;C, 1:10 dilution Perm/Wash buffer. Incubate at 4 &#176;C for 20&#8211;30 min to fully permeabilize the cells.</li>
		<li>Centrifuge at 600 x g at 4 &#176;C for 5 min and aspirate the supernatant.</li>
		<li>For intracellular staining, follow similar staining steps (as for surface staining). However, use the 4 &#176;C, 1:10 dilution Perm/Wash buffer instead of the CSM to make the total staining volume so that the cells remain in a permeabilizing environment throughout the intracellular staining.</li>
		<li>Add the intracellular antibody cocktail to the surface stained and permeabilized cell pellet (steps 2.1.2&#8211;2.1.7). Bring the total staining volume to 50 &#956;L per number of cell samples. Incubate for 60 min at 4 &#176;C. Agitate the sample every 15 min to ensure even staining.</li>
		<li>During the intracellular staining, prepare the intercalator solution: 900 &#956;L of filtered PBS + 100 &#956;L of filtered 16% PFA (final concentration of 1.6% PFA) + 0.2 &#956;L of 500 uM of intercalator dye (Table of Materials).</li>
		<li>Top off the cell pellet + intracellular antibody cocktail with cold 1:10 Perm/Wash buffer.</li>
		<li>Centrifuge at 600 x g at 4 &#176;C for 5 min, and aspirate the supernatant. Top off the staining tube with CSM. Count and record the cell number. Centrifuge at 600 x g at 4 &#176;C for 5 min, and aspirate the supernatant. Resuspend the cells in 1 mL of intercalator solution from step 4.9. Incubate for at least 20 min at room temperature for full intercalation or overnight at 4 &#176;C (samples in intercalator solution can stay at 4 &#176;C for up to 1 week before running them on the mass cytometry instrument).</li>
	</ol>
	</li>
	<li>Prepare the cells for the mass cytometry instrument.
	<ol>
		<li>Top off the tube with 3 mL of filtered ddH2O, and centrifuge at 600 x g for 5 min at 4 &#176;C. Resuspend the cells in 3 mL of filtered ddH2O. Pass this suspension through a 100 &#956;m filter to remove any debris or aggregates that could potentially clog the mass cytometry instrument.</li>
		<li>Count and record the cell number post-filtration. Centrifuge at 600 x g for 5 min at 4 &#176;C</li>
	</ol>
	</li>
	<li>Prepare the calibration bead solution (Table of Materials) by diluting it 1:10 in filtered ddH2O.</li>
	<li>Resuspend the stained cells in the required volume of 1:10 diluted calibration bead solution to attain a cell concentration of ~1 x 106 cells/mL. 
	Note: For a CyTOF1 at 45 &#181;L/min, the recommended optimum is 5 x 105 cells/mL; for Helios at 30 &#181;L/min, 7.5 x 105 cells/mL.</li>
	<li>Proceed to run the sample on the mass cytometry instrument.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ruxolitinib</td>
			<td>Santa Cruz</td>
			<td>SC-364729A</td>
			<td>Stock Conc: 10 mM; Final Conc: 5 &#956;M</td>
		</tr>
		<tr>
			<td>R848</td>
			<td>Invivogen</td>
			<td>tlrl-r848-5</td>
			<td>Stock Conc: 1 &#956;g/&#956;L; Final Conc: 1 &#956;g/mL</td>
		</tr>
		<tr>
			<td>LPS-EK</td>
			<td>Invivogen</td>
			<td>tlrl-eklps</td>
			<td>Stock Conc: 1 &#956;g/&#956;L; Final Conc: 0.1 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Sterile PBS</td>
			<td>Lonza</td>
			<td>17-516F</td>
			<td></td>
		</tr>
		<tr>
			<td>Lyse/Fix Buffer</td>
			<td>BD biosciences</td>
			<td>558049</td>
			<td>Stock Conc: 5X; Final Conc: 1X (dilute in ddH2O)</td>
		</tr>
		<tr>
			<td>BD Phosflow perm/wash buffer I</td>
			<td>BD biosciences</td>
			<td>557885</td>
			<td>Stock Conc: 10X; Final Conc: 1:10 (dilute in ddH2O)</td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Gibco</td>
			<td>21870-076</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Azide (NaN3)</td>
			<td>Sigma-Aldrich</td>
			<td>S-8032</td>
			<td>Stock Conc:&#62;99.9%; Final Conc: 0.0002</td>
		</tr>
		<tr>
			<td>Protein Transport Inhibitor (PTI)</td>
			<td>eBiosciences</td>
			<td>00-4980-93</td>
			<td>Stock Conc: 500X; Final Conc: 1X</td>
		</tr>
		<tr>
			<td>DNA Intercalator</td>
			<td>Fluidigm</td>
			<td>201192B</td>
			<td>Stock Conc: 500 &#956;M; Final Conc: 0.1 &#956;M</td>
		</tr>
		<tr>
			<td>MaxPar Barcode Perm Buffer</td>
			<td>Fluidigm</td>
			<td>201057</td>
			<td>Stock Conc: 10X; Final Conc: 1X</td>
		</tr>
		<tr>
			<td>20-plex Pd Barcode Set</td>
			<td>Fluidigm</td>
			<td>S0014</td>
			<td>Stock Conc: n/a; Final Conc: n/a</td>
		</tr>
		<tr>
			<td>Antibodies used for Mass Cytometry</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surface markers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CD1c</td>
			<td>Biolegend</td>
			<td>L161</td>
			<td>Mass: 161</td>
		</tr>
		<tr>
			<td>CD3</td>
			<td>BD</td>
			<td>UCHT1</td>
			<td>Mass: 144</td>
		</tr>
		<tr>
			<td>CD4</td>
			<td>Biolegend</td>
			<td>SK3</td>
			<td>Mass: 174</td>
		</tr>
		<tr>
			<td>CD7</td>
			<td>BD</td>
			<td>M-T701</td>
			<td>Mass: 149</td>
		</tr>
		<tr>
			<td>CD8</td>
			<td>Biolegend</td>
			<td>SK1</td>
			<td>Mass: 142</td>
		</tr>
		<tr>
			<td>CD11b</td>
			<td>Fluidigm</td>
			<td>ICRF44</td>
			<td>Mass: 209</td>
		</tr>
		<tr>
			<td>CD11c</td>
			<td>BD</td>
			<td>B-ly6</td>
			<td>Mass: 152</td>
		</tr>
		<tr>
			<td>CD15</td>
			<td>BD</td>
			<td>HI98</td>
			<td>Mass: 115</td>
		</tr>
		<tr>
			<td>CD14</td>
			<td>Biolegend</td>
			<td>M5E2</td>
			<td>Mass: 154</td>
		</tr>
		<tr>
			<td>CD16</td>
			<td>eBioscience/Thermo</td>
			<td>B73.1</td>
			<td>Mass: 165</td>
		</tr>
		<tr>
			<td>CD19</td>
			<td>Santa Cruz</td>
			<td>SJ25C1</td>
			<td>Mass: 163</td>
		</tr>
		<tr>
			<td>CD21</td>
			<td>Biolegend</td>
			<td>Bu32</td>
			<td>Mass: 141</td>
		</tr>
		<tr>
			<td>CD27</td>
			<td>BD</td>
			<td>L128</td>
			<td>Mass: 155</td>
		</tr>
		<tr>
			<td>CD38</td>
			<td>Fluidigm</td>
			<td>HIT2</td>
			<td>Mass: 172</td>
		</tr>
		<tr>
			<td>CD45 total</td>
			<td>Biolegend</td>
			<td>HI30</td>
			<td>Mass: 89</td>
		</tr>
		<tr>
			<td>CD45RA</td>
			<td>Biolegend</td>
			<td>HI100</td>
			<td>Mass: 153</td>
		</tr>
		<tr>
			<td>CD56</td>
			<td>Miltenyi</td>
			<td>REA196</td>
			<td>Mass: 168</td>
		</tr>
		<tr>
			<td>CD66</td>
			<td>BD</td>
			<td>B1.1/CD66</td>
			<td>Mass: 113</td>
		</tr>
		<tr>
			<td>CD86</td>
			<td>Fluidigm</td>
			<td>IT2.2</td>
			<td>Mass: 150</td>
		</tr>
		<tr>
			<td>CD123</td>
			<td>Fluidigm</td>
			<td>6H6</td>
			<td>Mass: 143</td>
		</tr>
		<tr>
			<td>CD278/ICOS</td>
			<td>Biolegend</td>
			<td>C398.4A</td>
			<td>Mass: 156</td>
		</tr>
		<tr>
			<td>CD179/PD1</td>
			<td>Biolegend</td>
			<td>EH12.2H7</td>
			<td>Mass: 162</td>
		</tr>
		<tr>
			<td>IgD</td>
			<td>Biolegend</td>
			<td>IA6-2</td>
			<td>Mass: 146</td>
		</tr>
		<tr>
			<td>IgM</td>
			<td>Biolegend</td>
			<td>MHM-88</td>
			<td>Mass: 151</td>
		</tr>
		<tr>
			<td>CXCR5</td>
			<td>BD</td>
			<td>RF8B2</td>
			<td>Mass: 173</td>
		</tr>
		<tr>
			<td>HLADR</td>
			<td>Biolegend</td>
			<td>L243</td>
			<td>Mass: 167</td>
		</tr>
		<tr>
			<td>Cytokines</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IL-1&#945;</td>
			<td>Biolegend</td>
			<td>364-3B3-14</td>
			<td>Mass: 147</td>
		</tr>
		<tr>
			<td>IL-1&#946;</td>
			<td>Biolegend</td>
			<td>H1b-98</td>
			<td>Mass: 169</td>
		</tr>
		<tr>
			<td>IL-1RA</td>
			<td>Santa Cruz</td>
			<td>AS17</td>
			<td>Mass: 157</td>
		</tr>
		<tr>
			<td>IL-6</td>
			<td>Biolegend</td>
			<td>MQ2-13A5</td>
			<td>Mass: 164</td>
		</tr>
		<tr>
			<td>IL-8</td>
			<td>BD</td>
			<td>E8N1</td>
			<td>Mass: 160</td>
		</tr>
		<tr>
			<td>IL-12/IL-23p40</td>
			<td>Biolegend</td>
			<td>C8.6</td>
			<td>Mass: 171</td>
		</tr>
		<tr>
			<td>IL-17A</td>
			<td>Biolegend</td>
			<td>BL168</td>
			<td>Mass: 148</td>
		</tr>
		<tr>
			<td>IL23p19</td>
			<td>eBioscience/Thermo</td>
			<td>23dcdp</td>
			<td>Mass: 176</td>
		</tr>
		<tr>
			<td>MIP1&#946;</td>
			<td>BD</td>
			<td>D21-1351</td>
			<td>Mass: 158</td>
		</tr>
		<tr>
			<td>MCP1</td>
			<td>BD</td>
			<td>5D3-F7</td>
			<td>Mass: 170</td>
		</tr>
		<tr>
			<td>IFN&#945;</td>
			<td>Miltenyi</td>
			<td>LT27:295</td>
			<td>Mass: 175</td>
		</tr>
		<tr>
			<td>IFN&#947;</td>
			<td>Biolegend</td>
			<td>4S.B3</td>
			<td>Mass: 165</td>
		</tr>
		<tr>
			<td>PTEN</td>
			<td>BD</td>
			<td>A2B1</td>
			<td>Mass: 159</td>
		</tr>
		<tr>
			<td>TNF&#945;</td>
			<td>Biolegend</td>
			<td>Mab11</td>
			<td>Mass: 166</td>
		</tr>
		<tr>
			<td>Note: If the manufacturer is stated as Fluidigm, this antibody was purchased from Fluidigm&#160;with metal pre-conjugated. If the manufacturer is stated as other than Fluidigm, this antibody was self-conjugated using the MaxPar Multi-Metal Labeling Kit (Fluidigm Cat: 201300) according to manufacturer protocol.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a barcoding assay for phenotypic and functional characterization of immune cells

Source: Baxter, R. M., et al. <a href="https://app.jove.com/v/57780">Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry.</a> J. Vis. Exp. (2018).
This video highlights a single-cell proteomic method that employs heavy-metal-conjugated antibodies for distinct cell barcoding. Subsequently, immunostaining and mass cytometry are applied to evaluate immune phenotypic and functional alterations in human whole blood-derived immune cells.

A Barcoding Assay for Phenotypic and Functional Characterization of Immune Cells

1. Barcode of Lysed/Fixed Blood Cells

	Thaw the lysed/fixed cell samples from the -80 &#176;C storage slowly on ice; a maximum of 20 samples can be ...

Begin with test samples containing stimulated and fixed immune cells.
Stimulation induces the expression of specific markers in cells, whereas fixation preserves phenotypic markers, including cell surface antigens, and functional state markers, including cytokines.
Add a unique combination of heavy metal isotope-conjugated antibodies to each sample.
This technique uniquely barcodes multiple samples, distinguishing each sample by its combination of heavy metal isotopes.
The antibodies bind to a common epitope, barcoding all the cells in the samples.
Combine all the barcoded samples in a single tube, for downstream processing.
Add a metal-conjugated antibody cocktail to stain surface antigens.
Add permeabilization buffer to increase cell membrane permeability for intracellular staining.
Add another set of metal-conjugated antibodies to stain intracellular cytokines.
Barcoding enables simultaneous staining and acquisition of the pooled samples via mass cytometry. This technique maximizes assay efficiency,&#160; improving the evaluation of phenotypic and functional state markers.

a-barcoding-assay-for-phenotypic-functional-characterization-immune

Research

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

70 Views. Source: Baxter, R. M., et al. Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry. J. Vis. Exp. (2018). This video highlights a single-cell proteomic method that employs heavy-metal-conjugated antibodies for distinct cell barcoding. Subsequently, immunostaining and mass cytometry are applied to evaluate immune phenotypic and functional alterations in human whole blood-derived immune cells. 

Video: A Barcoding Assay for Phenotypic and Functional Characterization of Immune Cells - Concept

Isolation and Staining of Mouse Skin Keratinocytes for Cell Cycle Specific Analysis of Cellular Protein Expression by Mass Cytometry

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the epidermis of mouse skin. There are seven important steps: separation of the epidermis from the dermis, digestion of the epidermis, staining of the epidermal cell populations with cisplatin, sample barcoding, staining with metal tagged antibodies for cell cycle markers and proteins of interest, detection of metal-tagged antibodies by mass cytometry, and the analysis of expression in the various cell cycle phases. The advantage of this approach over histological methods is the potential to assay the expression pattern of &#62;40 different markers in a single cell at different phases of the cell cycle. This approach also allows for the multivariate correlation analysis of protein expression that is more quantifiable than histological/imaging methods. The disadvantage of this protocol is that a suspension of single cells is needed, which results in the loss of location information provided by the staining of tissue sections. This approach may also require the inclusion of additional markers to identify different cell types in crude cell suspensions. The application of this protocol is evident in the analysis of hyperplastic skin disease models. Moreover, this protocol can be adapted for the analysis of specific sub-type of cells (e.g., stem cells) by the addition of lineage-specific antibodies. This protocol can also be adapted for the analysis of skin cells in other experimental species.

This protocol describes how to isolate skin keratinocytes from mouse models, to stain with metal-tagged antibodies, and to analyze stained cells by mass cytometry in order to profile the expression pattern of proteins of interest in the different cell cycle phases.

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the ...

JoVE Journal

Developmental Biology

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...

Immunology and Infection

Sample Preparation for Mass Cytometry Analysis

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities for deep analysis well beyond what is possible with conventional fluorescence-based flow cytometry. As with any new technology, there are critical steps that help ensure the reliable generation of high-quality data. Presented here is an optimized protocol that incorporates multiple techniques for the processing of cell samples for mass cytometry analysis. The methods described here will help the user avoid common pitfalls and achieve consistent results by minimizing variability, which can lead to inaccurate data. To inform experimental design, the rationale behind optional or alternative steps in the protocol and their efficacy in uncovering new findings in the biology of the system being investigated is covered. Lastly, representative data is presented to illustrate expected results from the techniques presented here.

This article describes the collection and processing of samples for mass cytometry analysis.

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities ...

A Barcoding Assay for Phenotypic and Functional Characterization of Immune Cells

Transcript

A Barcoding Assay for Phenotypic and Functional Characterization of Immune Cells

Isolation and Staining of Mouse Skin Keratinocytes for Cell Cycle Specific Analysis of Cellular Protein Expression by Mass Cytometry

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

Sample Preparation for Mass Cytometry Analysis