Part 1.  Instrumentation and Reagent Generation

An excellent introduction to the CyTOF platform is this video presentation from Suzanne Heck (University of Oxford). The video introduces the platform, sample preparation, and analysis approaches with examples.

Instrument start-up and QC resource(s):  

Start-up of Helios system

 

Panel Design

A successful CyTOF experiment depends on the success of the panel design. HIMC’s metal selection guide and Fluidigms guidelines pdf and mass cytometry panel design powerpoint, included below, are excellent resources to use when developing CyTOF panels.  References and user guides are also included below.  Briefly, a good working panel optimizes the signal for each marker while minimizing the noise/carryover from both metals and proteins.

Optimizing the signal requires in depth knowledge of mass metal intensity and protein expression in the field of study.  The low and high end of the mass window for metals has lower signal intensities compared to the middle end and therefore low expressing proteins should be paired with high signal intensity metals and vice versa.

Noise/Carryover consists of metal impurities that yield a “Mass+1” noise and oxidation in the argon plasma that yields a “Mass+16” carryover.  When considering which antibody to pair with a certain metal, M+1 and M+16 must always be investigated to make sure false positive signals do not exist. 

Steps for Panel Design

1) Make a list of protein markers to investigate

2) Rate the protein markers and metals from H, M, L in terms of expression level, H= highest, M= medium, L= lowest

•       Keep in mind: the biological setting the cells will be in to measure expression and also resolution and expression type for each protein.

•       Highest level expression should be conjugated to the lower metals compared to the lowest expression that should be on the higher metals with no major +1/+16 carry over

3) Account for all metal impurities and oxides

4) Pair antibodies to metals based off of steps 1-3

5) Titer and validate the whole panel on multiple subjects

6) Investigate false positive signals by

•       Staining the whole panel minus 1 for each metal/ab conjugate

•       Using positive and negative controls.

PDF guidelines for panel design

HIMC: Metal selection guidelines

Fluidigms guidelines for mass cytometry panel design

Fluidigm Maxpar Panel Designer user guide

Designing the Panel guidelines by cheeky scientist (Bushnell T, Expert.cheekyscientist.com)

Table of Isotopic Masses and Natural Abundances (Moltensalt.org)

Powerpoint presentation about panel design

Fluidigm: Mass Cytometry Panel Design

Peer review references

Compensation of Signal Spillover in Suspension and Imaging Mass Cytometry (Chevrier S, et. al., 2018, Cell Syst.)

Average Overlap Frequency: A simple metric to evaluate staining quality and community identification in high dimensional mass cytometry experiments (Amir EAD, et al. 2018, J Immunol Methods)

Mass Cytometry Panel Optimization Through the Designed Distribution of Signal Interference (Takahashi C, et. al., 2016, Cytometry A)

Evaluating the efficiency of isotope transmission for improved panel design and a comparison of the detection sensitivities of mass cytometer instruments (Tricot S, et. al., 2015, Cytometry A)

Normalization of mass cytometry data with bead standards (Finck R, et. al., 2013, Cytometry A)

A platinum-based covalent viability reagent for single cell mass cytometry (Fienberg H, et. al., 2012, Cytometry A)

Development of analytical methods for multiplex bio-assay with inductively coupled plasma mass spectrometry(Ornatsky O, et. al., 2008, J Anal At Spectrom)


Antibody conjugation resource(s), including testing/titration: 

Antibody conjugation for CyTOF is mostly standardized and follows an optimized protocol through Fluidigm.  Conjugating antibodies to metals allow users to customize and/or expand CyTOF panels and assures that the whole project can be stained with the same lot of validated antibody conjugated metals to reduce batch effects.

After conjugation, three very important procedures (protein concentration, validation, and titration) must be followed before using the antibody/metals on experimental samples.  1) Protein concentration (HIMC uses a nanodrop to read protein) of the conjugated antibody/metal must be determined BEFORE adding stabilizing solution.  If the protein readout is zero or very low, antibody was lost in a filtration step.  A positive protein readout only confirms presence of antibody and does not confirm conjugation.  2) Validation of the conjugated antibody/metal is performed by staining several samples at a higher concentration (between 10-15μg/ml) that are positive and negative for the antibody expression and running these samples on the CyTOF. If there is protein but no expression in the positive samples, most likely the conjugation failed, and that antibody must be re-conjugated.  3) Once the conjugated antibody/metal is validated (positive expression on the CyTOF), titration of the antibody/metal is established by analysis of serial dilution (between 0.5-20ug/ml) staining runs on the CyTOF.  Titration yields the best concentration not only for protein expression but also for minimal carryover.  After all antibodies are titrated, the whole panel should be used to stain several different samples to confirm the correct panel design.

A simplified workflow of antibody conjugation consists of two main steps.  Step 1) consists of parallel incubations of polymer/metal binding and antibody/TCEP for disulfide bond breakage.  After incubation, a quick wash step is required.  Step 2) consists of incubation for conjugation of antibody onto the metal/polymer.  Several washes with buffers help purify the antibody conjugated metal to make it ready to validate the conjugation and titer the antibody.  The Fluidigm protocol describes in detail conjugation with both MCP9(Cd metals) and X8(optimized for ICS) polymer and is linked below.  The DN3 (optimized for surface staining) polymer can be conjugated using the X8 protocol.

Antibody conjugation protocol

Fluidigm: MAXPAR ANTIBODY LABELING PROTOCOL.

Compatibility of Cadmium-Labeled Antibodies with Existing Protocols

Garry Nolan Lab protocols for pre-loading and carrier protein removal

Nolan Lab Metal Labeling IgG CyToF Antibodies with Maleimide MaxPar Reagents - Pre-Loading Method

Nolan lab: Antibody/BSA carrier protein removal protocol

Peer review references

Enabling Indium Channels for Mass Cytometry by Using Reinforced Cyclam-Based Chelating Polylysine (Grenier L. et. al., 2020, Bioconjug Chem.)

Labeling Antibodies Using Europium (Berg EA and Fishman JB, 2020, Cold Spring Harb Protoc.)

Method for Tagging Antibodies with Metals for Mass Cytometry Experiments (Chang SG and Guidos CJ, 2019, Book chapter in Methods in Molecular Biology)

Quantitative Measurement of Cell-Nanoparticle Interactions Using Mass Cytometry (Mitchell AJ. et. al., 2019, Methods Mol Biol.) 

Multiplex MHC Class I Tetramer Combined with Intranuclear Staining by Mass Cytometry (Simoni Y. et. al., 2019, Methods Mol Biol.)

Scalable Conjugation and Characterization of Immunoglobulins with Stable Mass Isotope Reporters for Single-Cell Mass Cytometry Analysis (Hartmann FJ. Et. al., 2019, Methods Mol Biol.)

Stabilizing Antibody Cocktails for Mass Cytometry (Schulz AR. et. al., 2019, Cytometry A)

Lanthanide nanoparticles for high sensitivity multiparameter single cell analysis (Pichaandi J. et. al., 2019, Chem Sci.) 

Metal-isotope-tagged monoclonal antibodies for high-dimensional mass cytometry (Han G. et. al., 2018, Nat Protoc.)

Aptamer-facilitated mass cytometry (Mironov GG. et. al., 2018, Anal Bioanal Chem)

Comparison of CyTOF assays across sites: Results of a six-center pilot study (Leipold MD. et. al., 2018, J Immunol Methods)

Atomic mass tag of bismuth-209 for increasing the immunoassay multiplexing capacity of mass cytometry (Han G. et. al., 2017, Cytometry A)

Dual-labelled antibodies for flow and mass cytometry: A new tool for cross-platform comparison and enrichment of target cells for mass cytometry (Baumgart S. et. al., 2017, Eur J Immunol.)

ICP-MS-based multiplex profiling of glycoproteins using lectins conjugated to lanthanide-chelating polymers (Leipold MD. et. al., 2009, J Proteome Res.)

Validating conjugations and viewing spillover for panel design

Youtube video about panel design

Establishing a robust 37-marker mass cytometry assay for deep single-cell immune profiling of whole blood (Rahman, A and Al-Maarri M, 2019, Youtube video)

Interactive website for antibody marker expression

Astrolabe diagnostics antibody staining data set to show expression levels in specific cells (Amir EAD et. al., 2019, Astrolabediagnostics.com)

Peer review reference

Osmium-Labeled Microspheres for Bead-Based Assays in Mass Cytometry (Budzinski L, et. al., 2019, J Immunol)

Optimization of Receptor Occupancy Assays in Mass Cytometry: Standardization Across Channels with QSC Beads (Bringeland GH. et. al., 2019, Cytometry A)

Unlabeled Competitor Antibody to Reduce Nonlinear Signal Spillover in Mass Cytometry (Sekhri P. et. al., 2019, Cytometry A)

Titration of Mass Cytometry Reagents (Vreden C. et. al., 2019, Methods Mol Biol.)

Titrating Complex Mass Cytometry Panels (Gullaksen SE. et. al., 2019, Cytometry A)

Sample barcoding:

In fluorescence flow cytometry, sample barcoding was first introduced by the Nolan lab as a method for reducing sample-to-sample variation, by labeling each individual sample with a unique combination of dyes and then combining them to stain and run as a composite sample.  Cells belonging to each individual sample are then retrieved during analysis by gating on the individual dye combinations, or “barcodes”.  This approach has multiple advantages, including:

  • Reduced variability from staining and acquisition, for the samples that are combined into a single composite sample.
  • Reduced antibody use, since the amount of antibody needed for a composite sample is usually less than that needed to stain all the samples individually (we usually see a 3-4x reduction in antibody requirement, but this should be tested for your cocktail and staining conditions).
  • Slightly reduced run time, since there is no need to remove one sample, rinse or backflush, and load the next sample.

These advantages can be realized for CyTOF as well, and are perhaps most important when making comparisons of intensity, where there is a need to robustly identify small shifts in markers like signaling proteins.  For CyTOF, barcoding strategies generally follow an “X of Y” scheme, where each barcode consists of X metal labels out of a possible Y metals reserved for barcoding.  For example, a “2 of 8” scheme would mean that every sample is labeled with a unique combination of 2 metals out of a possible 8.  Since there are 28 such combinations, this particular scheme allows for up to 28 samples to be combined in a single composite sample.  Such “X of Y” schemes have the added advantage that they allow for removal of cell doublets.  This is because any cell event that has more than X metal labels is by definition an aggregate of two or more different barcodes.

The first method for CyTOF barcoding was published by Behbehani et al., and used transient saponin permeabilization to introduce barcoding agents.  Since many staining protocols are optimized for live (not fixed) cells, methods using CD45 antibodies for live-cell barcoding have also been introduced (Mei et al., Lai et al.).  Finally, a “universal” live cell barcoding system for cells that may not express CD45 was described by Hartmann et al.  Deconvolution algorithms for identifying the single samples after acquisition have also been published (Zunder et al., Fread et al.).

Peer review references

Fluidigm: Cell-ID 20-Plex Pd Barcoding Kit

Platinum-conjugated antibodies for application in mass cytometry (Mei et al., Cytometry A, 2016)

Palladium-based mass tag cell barcoding with a doublet-filtering scheme and single-cell deconvolution algorithm (Zunder et al., Nature Protocol, 2015)

Live Cell Barcoding for Efficient Analysis of Small Samples by Mass Cytometry (Wagar, Methods in Molecular Biology, 2019)

Surface Barcoding of Live PBMC for Multiplexed Mass Cytometry (Schulz et al., Methods in Molecular Biology, 2019)

A Mass-Ratiometry-Based CD45 Barcoding Method for Mass Cytometry Detection (Meng et al., SLAS Technology, 2019)

Tellurium-based mass cytometry barcode for live and fixed cells (Willis et al., Cytometry A., 2018)

Automation of sample preparation for mass cytometry barcoding in support of clinical research: protocol optimization (Nassar et al., Anal Bioanal Chem, 2017)

Enhanced multiplexing in mass cytometry using osmium and ruthenium tetroxide species (Catena et al., Cytometry A., 2016)

Transient partial permeabilization with saponin enables cellular barcoding prior to surface marker staining (Behbehani et al., Cytometry A., 2014)

A CD45-based barcoding approach to multiplex mass-cytometry (CyTOF) (Lai et al., Cytometry A., 2015)

Minimizing Batch Effects in Mass Cytometry Data (Schuyler et al., Front. Immunol, 2019)

Ratiometric Barcoding for Mass Cytometry (Wu et al., Anal. Chem., 2018)

 

SuperSampler:  The SuperSampler is a third-party device for sample introduction on the CyTOF.  It has the advantage of accommodating up to a 50-ml sample tube, along with more effective intermittent sample resuspension, to aid in long sample runs.  See the video below.

For more information or inquiries about the SuperSampler, please refer to the following link:

http://www.victorianairships.com/

As for any experiment, controls in mass cytometry assays are most important. In the particular case of CyTOF, the controls will allow to assess:

1.     The reproducibility across the several batches of your experiment,

2.     The quality of stimulation and staining with internal controls included in given batch,

3.     The variability of CyTOF performance across the run of your samples.

Different types of controls can be used at once in your experiment:

·      Including an additional PBMC sample from a healthy individual. Buffy coats usually allow the isolation of several hundred thousand PBMCs that you can aliquot and freeze. Then you can thaw a vial per batch and use these cells as a repeated control across batches, with or without stimulation. One buffy coat should give you more than enough viable cells for use across several projects.

·      Veri-Cells™: Sold by Biolegend®, Veri-Cells™ for CyTOF are fixed and lyophilized PBMCs from healthy individual tagged with the heavy metal Tantalum (Ta). After reconstitution, you can directly spike them in your samples to a ratio 1:10. Then, you can proceed with your surface and intracellular staining as usual. The systematic use of Vericells helps you to determine the staining variability inter-experiment but also intra-experiment, as illustrated in the schematic below.

More detailed information on the use of Veri-Cells™ can be found on the Biolegend website and in this article: Sahaf B. et al., Methods Mol Biol., 2020.

·       EQ™ Four Element Calibration Beads from Fluidigm®. These are polystyrene bead standards containing known concentrations of the metal isotopes 140/142Ce, 151/153Eu, 165Ho, and 175/176Lu. We dilute the cells to the desired concentration with the 1X bead solution before running the samples on the CyTOF. We use the beads to normalize the CyTOF data for signal variation that occurs over time (instrument variability). Detailed information on the EQ calibration beads can be found here. This method is based on the work of Finck &al.

 

Continue to Part 2. CyTOF assays...