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Related Experiment Video

Updated: Aug 30, 2025

Automated Multiplex Immunofluorescence Panel for Immuno-oncology Studies on Formalin-fixed Carcinoma Tissue Specimens

Automated Multiplex Immunofluorescence Panel for Immuno-oncology Studies on Formalin-fixed Carcinoma Tissue Specimens

Published on: January 21, 2019

Mass cytometry immunostaining protocol for multiplexing clinical samples.

Ramy Gadalla¹, Giselle M Boukhaled², David G Brooks²

¹Princess Margaret Cancer Centre, University Health Network, Toronto, ON M5G 2M9, Canada.

|September 2, 2022

Summary

This summary is machine-generated.

This protocol uses DNA-based palladium barcoding before cell staining to multiplex up to 40 hematopoietic samples. This method reduces batch effects and antibody use for more reliable CyTOF analysis.

Keywords:

Flow Cytometry/Mass Cytometry Immunology

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Area of Science:

Immunology
Biotechnology
Analytical Chemistry

Background:

Cytometry by time-of-flight (CyTOF) enables high-parameter single-cell analysis.
Multiplexing samples in CyTOF reduces variability and increases throughput.
Existing CyTOF protocols can be affected by batch variations and high antibody consumption.

Purpose of the Study:

To develop an optimized CyTOF staining protocol for hematopoietic cells.
To implement a multiplexing strategy that minimizes batch effects and resource utilization.
To enhance the reproducibility and efficiency of CyTOF experiments.

Main Methods:

Development of a live-cell barcoding strategy using DNA-based palladium tags.
Integration of DNA barcoding prior to surface and intracellular immunostaining.
Application of receptor-type tyrosine-protein phosphatase C (CD45) antibodies conjugated to metal isotopes.

Main Results:

Successful multiplexing of up to 40 hematopoietic-derived cell samples.
Significant reduction in day-to-day staining and instrument sensitivity variations (batch effects).
Demonstrated decrease in antibody consumption and elimination of repeated instrument adjustments.

Conclusions:

The described CyTOF protocol effectively reduces batch effects through pre-staining DNA barcoding.
This method enhances sample multiplexing efficiency and lowers operational costs.
The protocol offers a robust and reproducible approach for high-parameter single-cell analysis of hematopoietic cells.