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Updated: Apr 18, 2026
Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester
Published on: June 4, 2019
Quantifying CD4 receptor protein in two human CD4+ lymphocyte preparations for quantitative flow cytometry.
Meiyao Wang1, Martin Misakian2, Hua-Jun He3
1Institute for Bioscience and Biotechnology Research, University of Maryland, 9600 Gudelsky Drive, Rockville, MD 20850 USA ; Biomolecular Measurement Division, National Institute of Standards and Technology (NIST), Gaithersburg, MD 20899 USA.
Lyophilized control cells show significantly lower CD4 density due to microvilli damage from the lyophilization process. This impacts their use as biological calibrators in flow cytometry, affecting CD4 protein expression measurements.
Area of Science:
- Immunology
- Biotechnology
- Analytical Chemistry
Background:
- Previous studies identified cryopreserved and lyophilized peripheral blood mononuclear cells (PBMC) as potential biological calibrators for quantitative flow cytometry.
- Lyophilized PBMC (Cyto-Trol™) exhibited ~16% lower CD4 protein expression than cryopreserved PBMC and fresh whole blood.
- Steric hindrance in anti-CD4 antibody binding to smaller lyophilized cells was hypothesized as a cause for lower expression.
Purpose of the Study:
- To quantify the CD4 receptor protein copy number per CD4+ lymphocyte using mass spectrometry.
- To investigate the structural differences in CD4+ lymphocytes between fresh, cryopreserved, and lyophilized preparations using electron microscopy.
- To elucidate the reasons for discrepancies in CD4 expression measurements between flow cytometry and mass spectrometry.
Main Methods:
- Targeted multiple reaction monitoring (MRM) mass spectrometry (MS) was employed to determine CD4 receptor copy number.
- Scanning electron microscopy (SEM) was used to examine cell morphology and surface structures.
Main Results:
- MRM MS determined CD4 copy numbers of (1.45 ± 0.09) × 10^5 and (0.85 ± 0.11) × 10^5 per CD4+ lymphocyte for cryopreserved PBMCs and lyophilized cells, respectively.
- Lyophilized cells showed greater variation in CD4 copy number across different signature peptides.
- SEM revealed significant morphological differences in CD4+ lymphocytes from lyophilized cells compared to fresh and cryopreserved samples.
Conclusions:
- The lyophilization process likely damages microvilli, leading to CD4 receptor loss and reduced CD4 density in lyophilized cells.
- Steric hindrance and potential CD4 receptor interactions with other biomolecules contribute to the lower CD4 density observed with flow cytometry compared to MRM MS.
