Name: mass cytometry: protocol for daily tuning and running cell samples on a cytof mass cytometer
Uploaded: 2012-11-02T14:00:00
Description: Watch this Scientific Journal Video about Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer at JoVE.com

We would like to thank Dr. Evan Newell and Dr. Sean Bendall for feedback. We would also like to thank DVS Sciences and Dr. Sean Bendall for a sample of the Multi-lanthanide-containing polystyrene beads. We are grateful for funding from NIH grant 2 U19 AI057229.

All cell samples for the CyTOF must be fixed and permeabilized. This enables greater entry of the iridium-containing DNA intercalator, and also prevents cell lysis during the MilliQ water wash and resuspension steps immediately before injecting into the mass cytometer.
1. Start-up of the Mass Cytometer
<ol>
 <li>Open the CyTOF software program. Select Instrument Setup. On the Card Cage tab, go to the lower right corner and click Heater "ON" button. Turn on the heater at least 15 min prior to...

<ol>
	<li>Bandura, 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=Mass+cytometry:+Technique+for+real+time+single+cell+multitarget+immunoassay+based+on+inductively+coupled+plasma+Time-of-Flight+mass+spectrometry.">Mass cytometry: Technique for real time single cell multitarget immunoassay based on inductively coupled plasma Time-of-Flight mass spectrometry.</a> Anal. Chem. 81, 6813-6822 (2009).</li><li>Lou, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymer-based+elemental+tags+for+sensitive+bioassays.">Polymer-based elemental tags for sensitive bioassays.</a> Angew. Chem., Int. Ed. 46, 6111-6114 (2007).</li><li>Majonis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Curious+results+with+palladium-+and+platinum-carrying+polymers+in+mass+cytometry+bioassays+and+an+unexpected+application+as+a+dead+cell+stain.">Curious results with palladium- and platinum-carrying polymers in mass cytometry bioassays and an unexpected application as a dead cell stain.</a> Biomacromolecules. 12, 3997-4010 (2011).</li><li>Leipold, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+mass+cytometry+methods+for+bacterial+discrimination.">Development of mass cytometry methods for bacterial discrimination.</a> Anal. Biochem. 419, 1-8 (2011).</li><li>Bendall, S. C. <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>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=Cytometry+by+Time-of-Flight+shows+combinatorial+cytokine+expression+and+virus-specific+cell+niches+within+a+continuum+of+CD8++T+cell+phenotypes.">Cytometry by Time-of-Flight shows combinatorial cytokine expression and virus-specific cell niches within a continuum of CD8+ T cell phenotypes.</a> Immunity. 36, 142-152 (2012).</li></ol>

For the last few decades, fluorescence flow cytometry has been a workhorse method for analyzing single cells, both in terms of surface expression and in functional assays. However, the issues of spectral overlap of the fluorescent dyes has limited the number of simultaneous markers. While experiments using more than 12 simultaneous markers have been reported, the amount of compensation necessary makes this technically difficult.
Instead of fluorophores, mass cytometry pioneered by DVS Sciences...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>CyTOF mass cytometer</td>
 <td>DVS Sciences</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Wash solution</td>
 <td>DVS Sciences</td>
 <td>201071</td>
 <td>0.05% hydrofluoric acid in water</td>
 </tr>
 <tr>
 <td>Tuning solution</td>
 <td>DVS Sciences</td>
 <td>201072</td>
 <td>0.5 ppm La, Cs, Tb, Tm, Ir, trace nitric acid</td>
 </tr>
 <tr>
 <td>Eu-containing polystyrene beads</td>
 <td>DVS Sciences</td>
 <td>201073</td>
 <td>Contains natural-abundance Eu isotopes; beads supplied at approximately 1 million/ml</td>
 </tr>
 <tr>
 <td>Multi-lanthanide-containing (La/Pr/Tb/Tm/Lu)- polystyrene beads</td>
 <td>DVS Sciences</td>
 <td>Not yet commercially-available</td>
 <td>Contains natural-abundance isotopes of listed lanthanides</td>
 </tr>
 <tr>
 <td>Nitric acid (concentrated)</td>
 <td>Fisher Scientific</td>
 <td>A467-500</td>
 <td>Optima trace-metal pure ICP-MS grade</td>
 </tr>
 <tr>
 <td>Polystyrene round-bottom tube with cell-strainer cap-5 ml</td>
 <td>BD</td>
 <td>352235</td>
 <td>used to filter cell samples before injection</td>
 </tr>
 <tr>
 <td>Norm-Ject tuberkulin syringe-1 ml</td>
 <td>Henke Sass Wolf</td>
 <td>4010-200V0</td>
 <td>silicone-free, latex-free</td>
 </tr>
 <tr>
 <td>Norm-Ject syringe-3 ml</td>
 <td>Henke Sass Wolf</td>
 <td>4010.000V0</td>
 <td>silicone-free, latex-free</td>
 </tr>
 <tr>
 <td>MilliQ water</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>18 M&Omega; pure water; must not be stored in glass or plastic bottles that have been washed with commercial detergent (due to their high levels of barium present).</td>
 </tr>
 <tr>
 <td>Citranox</td>
 <td>Sigma-Aldrich</td>
 <td>Z273236</td>
 <td>acid detergent</td>
 </tr>
 <tr>
 <td>Argon gas</td>
 <td>Praxair</td>
 <td>AR 5.0UH-T</td>
 <td>99.999% Ultra-high purity</td>
 </tr>
 </tbody>
</table>

mass cytometry: protocol for daily tuning and running cell samples on a cytof mass cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

Following the above protocol should accomplish four things. First, allowing adequate warm-up time for the mass cytometer will produce a hot, stable plasma necessary for optimal signal and minimal oxide formation. Second, adequate washing of the mass cytometer with MilliQ water and DVS Wash solution (Figure 9) will help reduce the levels of metal adsorption to the tubing and other parts of the machine, helping to reduce background during sample acquisition (Figure 8). It will also help re...

Watch this Scientific Journal Video about Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer at JoVE.com

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

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