Absolute Quantification of Plasma Membrane Receptors Via Quantitative Flow Cytometry

Fang, Yingye; Malik, Manasi; England, Sarah K.; Imoukhuede, P. I.

doi:10.1007/978-1-0716-2217-9_4

Cited by 6 publications

(8 citation statements)

References 34 publications

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“…2 A and B), titrating phycoerythrin (PE)-conjugated anti-HA antibody. We chose the PE-conjugated anti-HA antibody because PE antibody has a ﬂuorophore/protein ratio of 1.0, whereas other fluorophores (e.g., FITC and APC) are conjugated to antibodies at varying ratios [ 13 ]. Thirdly, we used Quantibrite PE calibration beads to create a calibration curve for the PE fluorescent signal, as previously described [ 13 ].…”

Section: Resultsmentioning

confidence: 99%

“…We chose the PE-conjugated anti-HA antibody because PE antibody has a ﬂuorophore/protein ratio of 1.0, whereas other fluorophores (e.g., FITC and APC) are conjugated to antibodies at varying ratios [ 13 ]. Thirdly, we used Quantibrite PE calibration beads to create a calibration curve for the PE fluorescent signal, as previously described [ 13 ]. At saturating concentrations of PE-conjugated anti-HA antibody (20 μg/mL for cell surface OXTR and 50 μg/mL for whole-cell OXTR), CRISPR-edited HMSMCs contained 4900 cell-surface OXTRs ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…To quantify OXTR protein expression on the cell surface, we prepared a single-cell suspension [ 13 ] and incubated the cells with saturating PE-conjugated anti-HA antibody and eFluor 780–conjugated fixable viability dye (FVD). To quantify total OXTR protein expression, we fixed and permeabilized the PE-labeled cells and incubated them again with PE-HA antibody to label the intracellular HA-OXTR ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Compared with the current gold standard assays for cell-surface protein measurements, such as western blotting and traditional flow cytometry, qFlow is a more quantitative tool for analyzing cell-surface and intracellular proteins. We previously used this approach to study cell-surface-bound receptors, such as receptor tyrosine kinases and G-protein–coupled receptors [ 13 ]. The quantitative single-cell receptor tyrosine kinase data revealed heterogeneity in tumor and tumor-associated vascular cells [ [19] , [20] , [21] ], vessel-like tubules [ 22 ], and normal vascular cells [ [23] , [24] , [25] , [26] ].…”

Section: Discussionmentioning

confidence: 99%

See 3 more Smart Citations

Quantification of surface-localized and total oxytocin receptor in myometrial smooth muscle cells

Fang,

Reinl,

Liu

et al. 2024

Heliyon

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 2 more Smart Citations

Quantification of surface-localized and total oxytocin receptor in myometrial smooth muscle cells

Fang,

Reinl,

Liu

et al. 2024

Heliyon

Self Cite

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“…Recent breakthroughs in qFlow have led to accurate, reproducible quantification and identification of differential receptor concentrations across normal and diseased cell phenotypes [17][18][19][20][21][22][23][24][25][26] . These advances enabled us to show, for the first time, how genetic variation affected the presentation of OXTR on the cell membrane.…”

Section: Precision Oxtr Modeling: the Power Of Quantitative Datamentioning

confidence: 99%

Understanding the effects of oxytocin receptor variants on OXT–OXT receptor binding: A mathematical model

Dubey,

Fang,

Tukei

et al. 2024

Preprint

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Approximately half of U.S. women giving birth annually receive Pitocin, the synthetic form of oxytocin (OXT), yet its effective dose can vary significantly, presenting safety concerns due to unpredictable responses, which may lead to adverse outcomes for both mother and baby. To address the need for improved dosing, we developed a data-driven mathematical model to predict OXT receptor (OXTR) binding. Our study focuses on five prevalent OXTR variants (V45L, P108A, L206V, V281M, and E339K) and their impact on OXT–OXTR binding dynamics in two distinct cell types: human embryonic kidney cells (HEK293T) and human myometrial smooth muscle cells. We parameterize the model with cell-specific OXTR surface localization measurements. To strengthen the robustness of our study, we conduct a comprehensive meta-analysis of OXT-OXTR binding, enabling parameterization of our model with cell-specific OXT-OXTR binding kinetics (myometrial OXT-OXTR Kd= 1.6 nM, kon= 6.8 × 105M−1min−1, and koff= 0.0011 min−1). Our meta-analysis reveals significant homogeneity in OXT-OXTR affinity across experiments and species with a Kd= 0.52-9.32 nM and mean Kd= 1.48 ± 0.51 nM. Our model achieves valuable several insights into designing dosage strategies. First, we predict that the OXTR complex reaches maximum occupancy at 10 nM OXT in myometrial cells and at 1 µM in HEK293T cells. This information is pivotal for guiding experimental design and data interpretation when working with these distinct cell types, emphasizing the need to consider cell-type-specific effects when choosing OXTR-transfected cell lines. Second, our model recapitulates the significant effects of genetic variants for both experimental and physiologically relevant systems, with V281M and E339K substantially compromising OXT–OXTR binding capacity. These findings suggest the need for personalized oxytocin dosing based on individual genetic profiles to enhance therapeutic efficacy and reduce risks, especially in the context of labor and delivery. Third, we demonstrate the potential for rescuing the attenuated cell response observed in V281M and E339K variants by increasing the OXT dosage at specific, early time points. Cellular responses to OXT, including Ca2+release, manifest within minutes. Our model indicates that providing V281M- and E339K-expressing cells with doubled OXT dose during the initial minute of binding can elevate OXT–OXTR complex formation to levels comparable to wild-type OXTR. In summary, our study provides a computational framework for precision oxytocin dosing strategies, paving the way for personalized medicine.

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Toward Blood-Based Precision Medicine: Identifying Age-Sex-Specific Vascular Biomarker Quantities on Circulating Vascular Cells

2023

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Introduction Abnormal angiogenesis is central to vascular disease and cancer, and noninvasive biomarkers of vascular origin are needed to evaluate patients and therapies. Vascular endothelial growth factor receptors (VEGFRs) are often dysregulated in these diseases, making them promising biomarkers, but the need for an invasive biopsy has limited biomarker research on VEGFRs. Here, we pioneer a blood biopsy approach to quantify VEGFR plasma membrane localization on two circulating vascular proxies: circulating endothelial cells (cECs) and circulating progenitor cells (cPCs). Methods Using quantitative flow cytometry, we examined VEGFR expression on cECs and cPCs in four age-sex groups: peri/premenopausal females (aged < 50 years), menopausal/postmenopausal females (≥ 50 years), and younger and older males with the same age cut-off (50 years). Results cECs in peri/premenopausal females consisted of two VEGFR populations: VEGFR-low (~ 55% of population: population medians ~ 3000 VEGFR1 and 3000 VEGFR2/cell) and VEGFR-high (~ 45%: 138,000 VEGFR1 and 39,000–236,000 VEGFR2/cell), while the menopausal/postmenopausal group only possessed the VEGFR-low cEC population; and 27% of cECs in males exhibited high plasma membrane VEGFR expression (206,000 VEGFR1 and 155,000 VEGFR2/cell). The absence of VEGFR-high cEC subpopulations in menopausal/postmenopausal females suggests that their high-VEGFR cECs are associated with menstruation and could be noninvasive proxies for studying the intersection of age-sex in angiogenesis. VEGFR1 plasma membrane localization in cPCs was detected only in menopausal/postmenopausal females, suggesting a menopause-specific regenerative mechanism. Conclusions Overall, our quantitative, noninvasive approach targeting cECs and cPCs has provided the first insights into how sex and age influence VEGFR plasma membrane localization in vascular cells.

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Absolute Quantification of Plasma Membrane Receptors Via Quantitative Flow Cytometry

Cited by 6 publications

References 34 publications

Quantification of surface-localized and total oxytocin receptor in myometrial smooth muscle cells

Quantification of surface-localized and total oxytocin receptor in myometrial smooth muscle cells

Understanding the effects of oxytocin receptor variants on OXT–OXT receptor binding: A mathematical model

Toward Blood-Based Precision Medicine: Identifying Age-Sex-Specific Vascular Biomarker Quantities on Circulating Vascular Cells

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