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Related Concept Videos

Flow Cytometry01:23

Flow Cytometry

The development of flow cytometry techniques began in 1934 with initial attempts by Andrew Moldavan, a bacteriologist who counted the cells in a flowing capillary system. Moldavan pumped cells through a capillary tube focused under a microscope for visualization. The invention of photometry allowed the measurement of differentially-stained cells, and Louis Kamentsky developed the first multiparameter flow cytometer in 1965 to identify and count the cancer cells in cervical tissue specimens.
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Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester
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Human CD4+ lymphocytes for antigen quantification: characterization using conventional flow cytometry and mass

Lili Wang1, Fatima Abbasi, Olga Ornatsky

  • 1Biochemical Science Division, National Institute of Standards and Technology, 100 Bureau Drive, Gaithersburg, MD 20899, USA. lili.wang@nist.gov

Cytometry. Part a : the Journal of the International Society for Analytical Cytology
|April 28, 2012
PubMed
Summary

Biological cell reference materials are crucial for standardizing antibody bound per cell (ABC) measurements. Cryopreserved peripheral blood mononuclear cells (PBMCs) provide consistent CD4 expression values, unlike some lyophilized preparations. Mass cytometry results are comparable to flow cytometry when standardized using the ABC approach.

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Area of Science:

  • Immunology
  • Biotechnology
  • Flow Cytometry
  • Mass Cytometry

Background:

  • Standardizing fluorescence intensity to antibody bound per cell (ABC) requires reliable biological cell reference materials.
  • Commercially available cryopreserved and lyophilized peripheral blood mononuclear cells (PBMCs) were evaluated for their suitability as reference materials.
  • CD4 expression was used as a reference biomarker to assess the comparability of different cell preparations.

Purpose of the Study:

  • To characterize cryopreserved and lyophilized PBMC preparations as potential biological reference materials for flow cytometry and mass cytometry.
  • To determine the antibody bound per cell (ABC) values for CD4 expression on different PBMC preparations.
  • To compare the performance of conventional flow cytometry and CyTOF™ mass cytometry using a standardized ABC approach.

Main Methods:

  • Peripheral blood mononuclear cells (PBMCs) were obtained fresh, cryopreserved, or from two lyophilized preparations (Cyto-Trol and PBMC-NIBSC).
  • Antibody bound per cell (ABC) values for CD4 expression were measured using conventional flow cytometry and CyTOF™ mass cytometry.
  • Simultaneous surface and intracellular staining, cell diameter measurements, and fixation effects were analyzed.

Main Results:

  • Cryopreserved PBMCs showed consistent CD4 ABC values comparable to fresh PBMCs and whole blood.
  • Lyophilized PBMCs (Cyto-Trol and PBMC-NIBSC) exhibited lower CD4 ABC values than fresh samples.
  • Paraformaldehyde fixation significantly reduced antibody binding sites on lyophilized PBMC-NIBSC, contributing to lower ABC values; smaller cell size may also cause steric hindrance.

Conclusions:

  • Cryopreserved PBMCs are suitable biological reference materials for standardizing antibody bound per cell (ABC) measurements.
  • Lyophilized PBMC preparations require careful consideration of fixation and potential steric effects for accurate quantification.
  • Standardized ABC approach confirms comparability between conventional flow cytometry and CyTOF™ mass cytometry.