Name: sample preparation for mass cytometry analysis
Uploaded: 2017-04-29T13:00:00
Description: Watch this Scientific Journal Video about Sample Preparation for Mass Cytometry Analysis at JoVE.com

Work in the Barton lab was supported by a grant from the Cancer Prevention and Research Institute of Texas (CPRIT RP110471). This research was supported in part, by a training grant fellowship for Ryan L. McCarthy from the National Institutes of Health Training Program in Molecular Genetics 5 T32 CA009299. The core facility that maintains and runs the mass cytometry machine is supported by CPRIT grant (RP121010) and NIH core grant (CA016672).

1. Cell Harvesting
<ol>
	<li>Harvesting cells from primary tissue 
	NOTE: The process described for harvesting cells from primary tissue is specifically applicable to mouse breast tumor tissue and may not be applicable as is to primary tissues from other sources.
	<ol>
		<li>Prepare digestion buffer by dissolving 5 mg hyaluronidase and 30 mg collagenase in 10 mL DMEM/F12 media per gram of tissue to be processed. Filter sterilize digestion buffer.</li>
		<li>Isolate primary mammary gland tumor from...

<ol>
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W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+tetramer+staining+and+mass+cytometry+analysis+facilitate+T-cell+epitope+mapping+and+characterization.">Combinatorial tetramer staining and mass cytometry analysis facilitate T-cell epitope mapping and characterization.</a> Nat Biotechnol. 31, 623-629 (2013).</li><li>Leong, M. L., Newell, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+Peptide-MHC+Tetramer+Staining+with+Mass+Cytometry.">Multiplexed Peptide-MHC Tetramer Staining with Mass Cytometry.</a> Methods Mol Biol. 1346, 115-131 (2015).</li><li>Zunder, E. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palladium-based+mass+tag+cell+barcoding+with+a+doublet-filtering+scheme+and+single-cell+deconvolution+algorithm.">Palladium-based mass tag cell barcoding with a doublet-filtering scheme and single-cell deconvolution algorithm.</a> Nat Protoc. 10, 316-333 (2015).</li><li>Lai, L., Ong, R., Li, J., Albani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+CD45-based+barcoding+approach+to+multiplex+mass-cytometry+(CyTOF).">A CD45-based barcoding approach to multiplex mass-cytometry (CyTOF).</a> Cytometry A. 87, 369-374 (2015).</li><li>Mei, H. E., Leipold, M. D., Schulz, A. R., Chester, C., Maecker, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Barcoding+of+live+human+peripheral+blood+mononuclear+cells+for+multiplexed+mass+cytometry.">Barcoding of live human peripheral blood mononuclear cells for multiplexed mass cytometry.</a> J Immunol. 194, 2022-2031 (2015).</li><li>Fienberg, H. G., Simonds, E. F., Fantl, W. J., Nolan, G. P., Bodenmiller, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+platinum-based+covalent+viability+reagent+for+single-cell+mass+cytometry.">A platinum-based covalent viability reagent for single-cell mass cytometry.</a> Cytometry A. 81, 467-475 (2012).</li><li>Krutzik, P. O., Nolan, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+cell+barcoding+in+flow+cytometry+allows+high-throughput+drug+screening+and+signaling+profiling.">Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling.</a> Nat Methods. 3, 361-368 (2006).</li><li>Behbehani, G. K., Bendall, S. C., Clutter, M. R., Fantl, W. J., Nolan, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+mass+cytometry+adapted+to+measurements+of+the+cell+cycle.">Single-cell mass cytometry adapted to measurements of the cell cycle.</a> Cytometry A. 81, 552-566 (2012).</li><li>Edgar, L. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+hypoxic+cells+using+an+organotellurium+tag+compatible+with+mass+cytometry.">Identification of hypoxic cells using an organotellurium tag compatible with mass cytometry.</a> Angew Chem Int Ed Engl. 53, 11473-11477 (2014).</li><li>Bendall, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+mass+cytometry+of+differential+immune+and+drug+responses+across+a+human+hematopoietic+continuum.">Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum.</a> Science. 332, 687-696 (2011).</li><li>Horowitz, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+environmental+determinants+of+human+NK+cell+diversity+revealed+by+mass+cytometry.">Genetic and environmental determinants of human NK cell diversity revealed by mass cytometry.</a> Sci Transl Med. 5, 208ra145 (2013).</li><li>Zunder, E. R., Lujan, E., Goltsev, Y., Wernig, M., Nolan, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+continuous+molecular+roadmap+to+iPSC+reprogramming+through+progression+analysis+of+single-cell+mass+cytometry.">A continuous molecular roadmap to iPSC reprogramming through progression analysis of single-cell mass cytometry.</a> Cell Stem Cell. 16, 323-337 (2015).</li><li>Han, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+mass+cytometry+reveals+intracellular+survival/proliferative+signaling+in+FLT3-ITD-mutated+AML+stem/progenitor+cells.">Single-cell mass cytometry reveals intracellular survival/proliferative signaling in FLT3-ITD-mutated AML stem/progenitor cells.</a> Cytometry A. 87, 346-356 (2015).</li><li>Bendall, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+trajectory+detection+uncovers+progression+and+regulatory+coordination+in+human+B+cell+development.">Single-cell trajectory detection uncovers progression and regulatory coordination in human B cell development.</a> Cell. 157, 714-725 (2014).</li><li>Qiu, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracting+a+cellular+hierarchy+from+high-dimensional+cytometry+data+with+SPADE.">Extracting a cellular hierarchy from high-dimensional cytometry data with SPADE.</a> Nat Biotechnol. 29, 886-891 (2011).</li><li>Amir el, A. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=viSNE+enables+visualization+of+high+dimensional+single-cell+data+and+reveals+phenotypic+heterogeneity+of+leukemia.">viSNE enables visualization of high dimensional single-cell data and reveals phenotypic heterogeneity of leukemia.</a> Nat Biotechnol. 31, 545-552 (2013).</li><li>Behbehani, G. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+partial+permeabilization+with+saponin+enables+cellular+barcoding+prior+to+surface+marker+staining.">Transient partial permeabilization with saponin enables cellular barcoding prior to surface marker staining.</a> Cytometry A. 85, 1011-1019 (2014).</li><li>Finck, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normalization+of+mass+cytometry+data+with+bead+standards.">Normalization of mass cytometry data with bead standards.</a> Cytometry A. 83, 483-494 (2013).</li><li>Chester, C., Maecker, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Algorithmic+Tools+for+Mining+High-Dimensional+Cytometry+Data.">Algorithmic Tools for Mining High-Dimensional Cytometry Data.</a> J Immunol. 195, 773-779 (2015).</li><li>Diggins, K. E., Ferrell, P. B., Irish, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+discovery+and+characterization+of+cell+subsets+in+high+dimensional+mass+cytometry+data.">Methods for discovery and characterization of cell subsets in high dimensional mass cytometry data.</a> Methods. 82, 55-63 (2015).</li><li>Leelatian, N., Diggins, K. E., Irish, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+Phenotypes+and+Signaling+Networks+of+Single+Human+Cells+by+Mass+Cytometry.">Characterizing Phenotypes and Signaling Networks of Single Human Cells by Mass Cytometry.</a> Methods Mol Biol. 1346, 99-113 (2015).</li><li>van der Maaten, L. J. P., Hinton, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizaing+Data+using+t-SNE.">Visualizaing Data using t-SNE.</a> J. Mach. Learn. Res. 9, 2431-2456 (2008).</li><li>Kling, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytometry:+Measure+for+measure.">Cytometry: Measure for measure.</a> Nature. 518, 439-443 (2015).</li><li>Gregori, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperspectral+cytometry.">Hyperspectral cytometry.</a> Curr Top Microbiol Immunol. 377, 191-210 (2014).</li><li>Chattopadhyay, P. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brilliant+violet+fluorophores:+a+new+class+of+ultrabright+fluorescent+compounds+for+immunofluorescence+experiments.">Brilliant violet fluorophores: a new class of ultrabright fluorescent compounds for immunofluorescence experiments.</a> Cytometry A. 81, 456-466 (2012).</li><li>Rieger, A. M., Havixbeck, J. J., Barreda, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X-FISH:+Analysis+of+cellular+RNA+expression+patterns+using+flow+cytometry.">X-FISH: Analysis of cellular RNA expression patterns using flow cytometry.</a> J Immunol Methods. 423, 111-119 (2015).</li><li>Frei, A. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+multiplexed+simultaneous+detection+of+RNAs+and+proteins+in+single+cells.">Highly multiplexed simultaneous detection of RNAs and proteins in single cells.</a> Nat Methods. 13, 269-275 (2016).</li></ol>

The protocol presented here has been successfully employed for the processing of various cultured cell lines (H1 and H9 hESCs, mESCs, MCF7, HEK 293, KBM5, HMEC, MCF10a) and primary tissue samples (mouse bone marrow, mouse embryonic liver, mouse adult liver, mouse tumor). Irrespective of the source, any tissue that can be dissociated into single cells while preserving the cellular state should be amenable to analysis by mass cytometry, but may require some protocol optimization. It is important to note that some epitopes ...

Cytometry enables the simultaneous measurement of multiple antibody targets at a single cell level across large populations of cells. In traditional fluorescence-based flow cytometry, the number of parameters that can be quantified is limited by spectral overlap between the emission spectrum of multiple fluorophores, which requires increasingly complex compensation calculations as the number of parameters increases. These limitations are addressed by mass cytometry, where heavy metal-conjugated antibodies are detected and quantified by time of flight (TOF) mass spectrometry to greatly expand the number of parameters collected simultaneously and yield a high dimensional protein-abundance profile for each individual cell.
A basic understanding of the workings of the mass cytometry instrument is beneficial to the user and may be essential for troubleshooting. For a more thorough description of tuning and running a mass cytometry machine, see the related manuscript1. Briefly, a cell sample is labeled with a panel of metal conjugated antibodies targeting cell surface markers, cytoplasmic proteins, nuclear proteins, chromatin-bound proteins or other epitopes of interest (Figure 1i). The labeled cells are loaded onto the machine, either one at a time by manual injection or using an autosampler that samples from a 96-well plate. The loaded cells are injected through a nebulizer, which generates a spray of liquid droplets encapsulating the cells (Figure 1ii). This spray is positioned so that the cells are ionized by an argon plasma torch. This ionization generates a particle cloud comprised of all the constituent atoms of each cell (Figure 1iii). As this particle cloud travels to the detector, low atomic mass atoms are separated from the high mass ions by a quadrupole mass filter. The remaining high mass ions continue to the detector, where the abundance of each isotope is quantified (Figure 1iv). The raw data collected by the detector, are analyzed by the mass cytometry instrument software to identify cell events. For each identified cell event, the signal detected in each channel is quantified and saved to an output .fcs file. The mass channels used for detecting the heavy metal conjugated antibodies exhibit minimal spectral overlap, which ranges from 0-4% with most contributions below 1%. Because of this low cross-talk between channels, it is generally unnecessary to transform the data with a compensation matrix before analysis2. However, care should be taken during the design of the experimental panel of antibodies to ensure that an antibody with a high-abundance epitope is not assigned to a mass channel that contributes signal to a channel assigned to a low-abundance antibody, as this may create an artificially high population in the channel receiving the signal contribution.
The exact preparation of samples for mass cytometry analysis is highly dependent upon the types of samples being collected and the experimental hypothesis being tested. We provide examples of two protocols for harvesting different types of cells (human embryonic stem cells) and primary cells (mouse breast tumor epithelial cell isolate). When beginning a mass cytometry study, it is advisable to first carry out a small pilot experiment to ensure that all protocols and antibodies to be utilized are working well in the investigator&#39;s hands. This is especially essential when custom conjugated antibodies are to be used, as the partial reduction reaction during the antibody conjugation has the potential to disrupt antibody affinity; therefore, each custom antibody needs to be validated empirically to ensure data accuracy. Other considerations include determining if cell surface antibodies to be used in the assay will recognize their epitopes after fixation or if they need to be applied to live cells. Some fixation may prevent proper staining with cell surface antibodies as is known to occur with peptide-MHC staining3,4. Performing cell surface antibody staining on live cells may be necessary for some antibodies, but this precludes the option of barcoding prior to antibody labeling as most barcoding methods are performed after fixation5 although antibody-based barcoding6,7 is an exception.
When determining the number of cells to be prepared for analysis, it is important to know that only a fraction of the cells loaded onto the machine will be detected and produce data. This loss is primarily due to inefficiencies when the nebulizer sprays the sample at the torch. The exact percent loss depends upon the mass cytometry machine setup and cell type but can range from 50-85% and should be considered when designing the experiment. This protocol has been optimized for 2x106 cells per sample but can be adapted for processing smaller or larger sample sizes. This protocol can be used with minimal modifications for sample sizes from 1 x 106 to 4 x 106, however it is critical that sample size be kept consistent within an experiment. Changes in cell number can affect the intensity of barcoding and antibody staining and should be kept roughly consistent across samples to be directly compared.
Some procedures, such as dead cell labeling and DNA labeling, should be carried out in the vast majority, if not all experimental designs. The labeling of dead cells in experiments analyzing only surface markers can be achieved using Rh103, but Rh103 should be avoided for experiments where permeabilization is required as it results in staining loss. For experiments involving permeabilization, it is advisable to utilize cisplatin8 and, when possible, an antibody recognizing cleaved PARP or cleaved Caspase to detect apoptotic and dead cells.
Barcoding samples can reduce antibody consumption, decrease acquisition time and eliminate sample-to-sample variability9. The process of barcoding involves applying a unique sample-specific code to all cells, which is used to assign each cell to its sample of origin. After barcoding the samples may be pooled prior to antibody staining, a process that ensures all samples are stained equally. To minimize experimental error, it is prudent to barcode and pool any samples that are to be directly compared to each other. Currently there exist several methods for barcoding samples for mass cytometry analysis5,6,7, two of which are presented here (see below).
With currently available reagents it is possible to quantify the abundance of 38 antibody targets, identify cells in S-phase10, distinguish live and dead cells8 and measure cellular levels of hypoxia11 in a multiplexed experiment of multiple samples. This ability to collect high parameter single cell data for large cell populations enables improved profiling of highly heterogeneous cell populations and has already produced a number of novel biological insights12,13,14,15,16. Distilling the resulting complex data to interpretable information has required the use of computational algorithms including Spanning Tree Progression of Density Normalized Events (SPADE)17 and viSNE18.
This article presents an accessible overview of how to design and carry out a mass cytometry experiment and introduces basic analysis of mass cytometry data.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>mTeSR1 medium kit</td>
			<td>Stem Cell technologies</td>
			<td>05850</td>
			<td>Warm at room temperature before use</td>
		</tr>
		<tr>
			<td>DMEM F-12</td>
			<td>ThermoFisher</td>
			<td>11330-032</td>
			<td>Warm at 37&#176;C before use</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Stem Cell technologies</td>
			<td>07920</td>
			<td>Warm at 37&#176;C before use</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Equitech</td>
			<td>BAH62</td>
			<td></td>
		</tr>
		<tr>
			<td>phosphate buffered saline</td>
			<td>Hyclone</td>
			<td>SH30256.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Sigma-Aldrich</td>
			<td>47036</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell ID Pt194</td>
			<td>Fluidigm</td>
			<td></td>
			<td>Provided at 1mM</td>
		</tr>
		<tr>
			<td>Cell ID Pt195</td>
			<td>Fluidigm</td>
			<td></td>
			<td>Provided at 1mM</td>
		</tr>
		<tr>
			<td>Cell ID Pt196</td>
			<td>Fluidigm</td>
			<td></td>
			<td>Provided at 1mM</td>
		</tr>
		<tr>
			<td>Cisplatin</td>
			<td>Enzo Life Sciences</td>
			<td>ALX-400-040-M250</td>
			<td>Soluble to 25mg/ml in DMSO</td>
		</tr>
		<tr>
			<td>Cell-ID 20-plex Pd Barcoding Kit</td>
			<td>Fluidigm</td>
			<td>201060</td>
			<td></td>
		</tr>
		<tr>
			<td>5-Iodo-2&#39;-deoxyuridine</td>
			<td>Sigma-Aldrich</td>
			<td>I7125</td>
			<td>Soluble to 74mg/ml in 0.2N NaOH</td>
		</tr>
		<tr>
			<td>Rhodium 103 intercelating agent</td>
			<td>Fluidigm</td>
			<td>201103A</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher</td>
			<td>BP1105-4</td>
			<td>Chill at -20&#176;C before use</td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>Sigma-Aldrich</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>5ml round bottom tubes</td>
			<td>Falcon</td>
			<td>352058</td>
			<td></td>
		</tr>
		<tr>
			<td>5ml 35um filter cap tubes</td>
			<td>Falcon</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>EQ four element calibration beads</td>
			<td>Fluidigm</td>
			<td>201078</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyaluronidase Type I-S</td>
			<td>Sigma-Aldrich</td>
			<td>H3506</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase from Clostridium histolyticum</td>
			<td>Sigma-Aldrich</td>
			<td>C9891</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Scalpel #10</td>
			<td>Sigma-Aldrich</td>
			<td>Z69239</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The protocol presented here can be broadly applied to a wide variety of cultured and primary cell samples with only minor modifications. Depending upon the requirements of the experiment, it can be performed in a modular manner when certain elements, such as barcoding or cell surface staining, are not necessary. Utilized in its entirety this protocol enables the preparation of multiplexed mass cytometry samples labeled for dead cell exclusion, S-phase population identification and stained...

Watch this Scientific Journal Video about Sample Preparation for Mass Cytometry Analysis at JoVE.com

Sample Preparation for Mass Cytometry Analysis

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 ...

The overall goal of this sample preparation process is to produce a robust and consistent antibody staining of diverse samples to enable the interrogation of heterogeneous cell populations. This method can help answer questions about cell identity, cell signaling and cell response in a complex heterogeneous cell population. The main advantage of this technique is it allows the measurement of high parameter protein levels at the single cell level.Demonstrating this procedure will be Aundrietta Duncan, a graduate student from our laboratory. For each single cell suspension sample, re-suspend it at four times 10 to the sixth cells per milliliter in serum free medium, add 500 microliters of freshly prepared 50 MicroMolar Cisplatin per 2/10 into the six cells, and incubate the samples on an orbital shaker with constant mixing at room temperature. After one minute, quench the Cisplatin with an equal volume of wash buffer and centrifuge the cells.Discard the supernatant and wash the samples two more times by adding 500 microliters of wash buffer, centrifuging the cells, and pouring off the wash buffer. Follow this by two washes in 500 microliters of PBS similarly centrifuging the samples and discarding the supernatant. After the second PBS wash, re-suspend the pellets in 500 microliters of fresh PBS and fix the cells with 500 microliters of 4%PFA.Pipette the cells to mix. Then place the samples on the orbital shaker with constant mixing for 15 minutes at room temperature. At the end of the incubation, pellet the cells by centrifugation, pour off the supernatant, and follow this by a wash in fresh PBS.To permeabilize the cells, spin down the cells again and discard the supernatants. Re-suspend the samples in the residual PBS by gentle vortexing, and immediately add one milliliter of 100%methanol per 2/10 10 to the sixth cells with gentle pipetting. Incubate the samples at four degrees for at least one hour, and at the end of the incubation, centrifuge the cells.Afterwards, pour off the methanol. Then wash the pellets in one milliliter of wash buffer for each milliliter of methanol, followed by two more washes in 500 microliters of fresh wash buffer. Using a 200 microliter pipette set to 50 microliters, transfer the remaining wash buffer from each pellet to a tube of the appropriate corresponding antibody cocktail.Pipette back up the combined solution without drawing air into the tip by keeping the plunger partially depressed. Then transfer the pipette tip into a 1.5 milliliter microcentrifuge tube containing 500 microliters of wash buffer without releasing the plunger. Release the plunger, drawing up the wash buffer for a total antibody cocktail volume of 50 microliters and label the appropriate corresponding sample with the aspirated antibody cocktail by gentle pipetting.After one hour at room temperature with constant shaking, wash the samples four times, pouring off the wash buffer each time. To label the cells with iridium, re-suspend the final pellets in 500 microliters of PBS, followed by the addition of 500 microliters of four percent PFA for 15 minutes at room temperature. Next, add 500 microliters of freshly prepared 62.5 nanomolar iridium and PFA to the samples for another 15 minutes of room temperature shaking.Then, centrifuge the cells, pour off the supernatant, and follow by two washes in 500 microliters of 0.1 percent BSA and filtered deionized water. Analyze the samples by adding normalization beads into the wells of sample plates, pipetting cells into these wells and then performing mass cytometry within 24 hours. Following normalization of the signal intensity, the calibration beads can be removed from the data by gating on the iridium 191 positive bead negative population.The single cell events can then be gated to remove the debris and cell multiplets using a biaxial plot of iridium 193 negative expression versus the event length. Cisplatin labeling can be used to measure the amount of cell depth. For example, here samples with low and higher quantities of dead cells are shown.The dead cells can be removed by gating away the platinum 198 positive population, barcoding results in a distinct separation of the samples within the barcoding parameters, allowing the cells to be assigned to their original sample identities. IDU labeling can be used to detect US face cells observed here. Following the initial normalization, de-barcoding and gating, the high dimensional data can be analyzed using the SPADE algorithm to generate a lower dimensional representation of the data that is more amenable to visual interpretation.Once mastered, this technique can be completed within four to six hours if performed properly. While attempting this procedure, it's important to remember to minimize sample loss while doing the wash step. After watching this video, you should have a good understanding of how to prepare samples for mass cytometry analysis.Don't forget that working with sodium azide can be hazardous, therefore you should use precautions such as wearing proper PPE when performing this procedure.

Research

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Biology

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