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Relative quantification of beta-adrenergic receptor in peripheral blood cells using flow cytometry.

ABSTRACT: Beta-adrenergic receptors (?-ARs) play a critical role in many diseases. Quantification of ?-AR density may have clinical implications in terms of assessing disease severity and identifying patients who could potentially benefit from beta-blocker therapy. Classical methods for ?-AR quantification are based on labor-intensive and time-consuming radioligand binding assays. Here, we report optimization of a flow cytometry-based method utilizing a biotinylated ?-AR ligand alprenolol as a probe and use of this method to quantify relative receptor expression in healthy controls (HC). Quantum™ MESF beads were used for quantification in absolute fluorescence units. The probe was chemically modified by adding a spacer moiety between biotin and alprenolol to stabilize receptor binding, thus preventing binding decay. Testing of three different standard cell fixation and permeabilization methods (formaldehyde fixation and saponin, Tween-20, or Triton-X 100 permeabilization) showed that the formaldehyde/Triton-X 100 method yielded the best results. ?-AR expression was significantly higher in granulocytes compared to mononuclear cells. These data show that flow cytometric quantification of relative ?-AR expression in circulating leukocytes is a suitable technology for large-scale clinical application. © 2018 International Society for Advancement of Cytometry.

SUBMITTER: Saygin D

PROVIDER: S-EPMC7263462 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Relative quantification of beta-adrenergic receptor in peripheral blood cells using flow cytometry.

Saygin Didem D Wanner Nicholas N Rose Jonathan A JA Naga Prasad Sathyamangla V SV Tang W H Wilson WHW Erzurum Serpil S Asosingh Kewal K

Cytometry. Part A : the journal of the International Society for Analytical Cytology 20180324 5

Beta-adrenergic receptors (β-ARs) play a critical role in many diseases. Quantification of β-AR density may have clinical implications in terms of assessing disease severity and identifying patients who could potentially benefit from beta-blocker therapy. Classical methods for β-AR quantification are based on labor-intensive and time-consuming radioligand binding assays. Here, we report optimization of a flow cytometry-based method utilizing a biotinylated β-AR ligand alprenolol as a probe and u ...[more]

PMID: 29573550

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Project description:Human peripheral blood mononuclear cells (PBMCs) originate from hematopoietic stem cells in the bone marrow, which mainly includes lymphocytes (T cells, B cells, and natural killer cells) and monocytes. Cryopreserved PBMCs providing biobank resources are crucial for clinical application or scientific research. Here, we used flow cytometry to explore the influence of long-term cryopreservation on the quality of PBMCs with the aim of providing important evidence for the effective utilization of biobank resources. The PBMCs were isolated from the peripheral blood, which was collected from volunteers in the hospital. After long-term cryopreservation in liquid nitrogen, we analyzed the changes in cell numbers, viability, and multiple subtypes of PBMCs and studied the apoptosis, proliferation, activation, function, and status of T cells in comparison with freshly isolated PBMCs by flow cytometry, and then further tracked the effects of long-term cryopreservation on the same sample. Although the different cell types in the PBMCs dynamically changed compared with those in the freshly isolated samples, PBMC recovery and viability remained stable after long-term cryopreservation, and the number of most innate immune cells (e.g., monocytes and B cells) was significantly reduced compared to that of the freshly isolated PBMCs or long-term cryopreserved PBMCs; more importantly, the proportion of T cell subtypes, apoptosis, proliferation, and functional T cells, except for Tregs, were not affected by long-term cryopreservation. However, the proportions of activated T, naïve T, central memory T, effector T, and effector memory T cells dynamically changed after long-term cryopreservation. This article provides important evidence for the effective utilization of biobank resources. Long-term cryopreserved PBMCs can be partly used as biological resources for clinical research or basic studies, but the effect of cryopreservation on PBMCs should be considered when selecting cell samples, especially in research relating to activating or inhibiting function.

Dataset Information

Relative quantification of beta-adrenergic receptor in peripheral blood cells using flow cytometry.

Publications

Relative quantification of beta-adrenergic receptor in peripheral blood cells using flow cytometry.

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