Curious Results with Palladium- and Platinum-Carrying Polymers in Mass Cytometry Bioassays and an Unexpected Application as a Dead Cell Stain

Majonis, Daniel; Ornatsky, Olga; Kinach, Robert; Winnik, Mitchell A.

doi:10.1021/bm201011t

Cited by 47 publications

(70 citation statements)

References 35 publications

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“…Different from a previous approach to generate Pd-labeled Ab (17), we utilized isothiocyanobenzyl-EDTA (SCN-Bn-EDTA), an amine-reactive compound carrying EDTA as a metal ion chelating moiety, to generate a series of CD45 antibodies labeled with highly isotopically pure palladium ions (Pd104, Pd106, Pd108, Pd110) (Figure 1A). SCN-Bn-EDTA was loaded with Pd ions in aqueous solution at 0.5 equivalents, lyophilized, and redissolved in DMSO for long-term storage at −20°C (14, 16).…”

Section: Resultsmentioning

confidence: 99%

“…Pd- and In-labeled CD45 antibodies were used over weeks/months without notable loss of staining intensity and were kept in aqueous buffer at 4°C, allowing for easier reagent handling as compared to hydrolysis-sensitive maleimido-mono-amide-DOTA compounds used previously (13). A previously described protocol to label Ab with Pd appeared to be more time consuming and difficult to perform in a lab not set up for chemical syntheses (17). In contrast, the protocol described herein to generate barcoding Ab is comparably simple and can be performed within two days, using commercially available materials and predominantly standard laboratory equipment.…”

Section: Discussionmentioning

confidence: 99%

“…In contrast to a previous approach to label Ab with Pd that led to reagents that stain dead cells (17), we used isothiocyanobenzyl-EDTA (SCN-Bn-EDTA) to achieve labeling of Ab with Pd (14, 16). …”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Barcoding of Live Human Peripheral Blood Mononuclear Cells for Multiplexed Mass Cytometry

Mei

Maecker

Schulz

et al. 2015

The Journal of Immunology

154

194

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Mass cytometry is developing as a means of multiparametric single cell analysis. Here, we present an approach to barcoding separate live human PBMC samples for combined preparation and acquisition on a CyTOF® instrument. Using six different anti-CD45 antibody (Ab) conjugates labeled with Pd104, Pd106, Pd108, Pd110, In113, and In115, respectively, we barcoded up to 20 samples with unique combinations of exactly three different CD45 Ab tags. Cell events carrying more than or less than three different tags were excluded from analyses during Boolean data deconvolution, allowing for precise sample assignment and the electronic removal of cell aggregates. Data from barcoded samples matched data from corresponding individually stained and acquired samples, at cell event recoveries similar to individual sample analyses. The approach greatly reduced technical noise and minimizes unwanted cell doublet events in mass cytometry data, and reduces wet work and antibody consumption. It also eliminates sample-to-sample carryover and the requirement of instrument cleaning between samples, thereby effectively reducing overall instrument runtime. Hence, CD45-barcoding facilitates accuracy of mass cytometric immunophenotyping studies, thus supporting biomarker discovery efforts, and should be applicable to fluorescence flow cytometry as well.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Barcoding of Live Human Peripheral Blood Mononuclear Cells for Multiplexed Mass Cytometry

Mei

Maecker

Schulz

et al. 2015

The Journal of Immunology

154

194

View full text Add to dashboard Cite

show abstract

“…Since palladium is not compatible with the DTPA-based polymer used for antibody labeling, it is not used for mass cytometry antibody measurements 9 . Palladium has six stable isotopes with masses of 102, 104, 105, 106, 108, and 110 u, which fall well below the 139–176 u mass range of the lanthanides, and therefore its use in MCB reagents does not impinge upon lanthanide-based antibody detection.…”

Section: Introductionmentioning

confidence: 99%

Palladium-based mass tag cell barcoding with a doublet-filtering scheme and single-cell deconvolution algorithm

et al. 2015

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SUMMARY Mass-tag cell barcoding (MCB) labels individual cell samples with unique combinatorial barcodes, after which they are pooled for processing and measurement as a single multiplexed sample. The MCB method eliminates variability between samples in antibody staining and instrument sensitivity, reduces antibody consumption, and shortens instrument measurement time. Here, we present an optimized MCB protocol with several improvements over previously described methods. The use of palladium-based labeling reagents expands the number of measurement channels available for mass cytometry and reduces interference with lanthanide-based antibody measurement. An error-detecting combinatorial barcoding scheme allows cell doublets to be identified and removed from the analysis. A debarcoding algorithm that is single cell-based rather than population-based improves the accuracy and efficiency of sample deconvolution. This debarcoding algorithm has been packaged into software that allows rapid and unbiased sample deconvolution. The MCB procedure takes 3–4 h, not including sample acquisition time of ~1 h per million cells.

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“…[2][3][4][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.…”

Section: Introductionmentioning

confidence: 99%

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

Maecker

2012

JoVE

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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][3][4][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. Video LinkThe video component of this article can be found at http://www.jove.com/video/4398/ ProtocolAll 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.

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Curious Results with Palladium- and Platinum-Carrying Polymers in Mass Cytometry Bioassays and an Unexpected Application as a Dead Cell Stain

Cited by 47 publications

References 35 publications

Barcoding of Live Human Peripheral Blood Mononuclear Cells for Multiplexed Mass Cytometry

Barcoding of Live Human Peripheral Blood Mononuclear Cells for Multiplexed Mass Cytometry

Palladium-based mass tag cell barcoding with a doublet-filtering scheme and single-cell deconvolution algorithm

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

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