A comparison of DNA methylation specific droplet digital PCR (ddPCR) and real time qPCR with flow cytometry in characterizing human T cells in peripheral blood

Abstract Background: CD137 is identified as a target for tumor immunotherapy. However, the role of CD137 in gastric cancer (GC), especially in inducing GC cell apoptosis has not been studied yet. Methods: Foxp3+ and CD8+ T cells in GCs were investigated by immunohistochemistry (IHC). CD137 expression in GCs was detected by flow cytometry, IHC and immunofluorescence (IF). Peripheral blood mononuclear cells (PBMCs) and CD8+ T cells isolated from peripheral blood were stimulated with a CD137 agonist in vitro. CD8+ T cells proliferation and p65 expression were explored by flow cytometry. p65 nuclear translocation was analyzed by IF. IL-10, TGF-β, IFN-γ, Perforin and Granzyme B were detected by real-time quantitative PCR (real-time PCR). PBMCs and primary GC cells were cocultured and stimulated with the CD137 agonist in vitro. Apoptosis of the primary GC cells was detected by flow cytometry. Results: Our data demonstrated that GC tumors show characteristics of an immunosuppressive microenvironment. CD137 was predominantly expressed in CD8+ T cells in GCs and had a positive correlation with tumor cell differentiation. CD137 agonist promoted CD8+ T cells proliferation and increased the secretion of IFN-γ, Perforin and Granzyme B, which induced primary GC cell apoptosis. Mechanistically, this study found that CD137 agonist could induce NF-κB nuclear translocation in CD8+ T cells. Conclusion: Our results demonstrate that CD137 agonist can induce primary GC cell apoptosis by enhancing CD8+ T cells via activating NF-κB signaling.

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Isolation and characterisation of antigen experienced CD4+ CD45RA+ CD31 - human T cells from normal human peripheral blood

Immunology Letters ◽

10.1016/s0165-2478(97)87627-4 ◽

1997 ◽

Vol 56 (1-3) ◽

pp. 197

Author(s):

A Thiel

Keyword(s):

T Cells ◽

Peripheral Blood ◽

Human Peripheral Blood ◽

Human T Cells ◽

Normal Human ◽

Normal Human Peripheral Blood

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Development and validation of a method for quantification of common wheat, durum wheat, rye and barley by droplet digital PCR

European Food Research and Technology ◽

10.1007/s00217-021-03786-y ◽

2021 ◽

Author(s):

Christian Schulze ◽

Anne-Catrin Geuthner ◽

Dietrich Mäde

Keyword(s):

Durum Wheat ◽

Real Time ◽

Bread Wheat ◽

Common Wheat ◽

Real Time Pcr ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Food Fraud ◽

Single Genome ◽

Cereal Species

AbstractFood fraud is becoming a prominent topic in the food industry. Thus, valid methods for detecting potential adulterations are necessary to identify instances of food fraud in cereal products, a significant component of human diet. In this work, primer–probe systems for real-time PCR and droplet digital PCR (ddPCR) for the detection of these cereal species: bread wheat (together with spelt), durum wheat, rye and barley for real-time PCR and ddPCR were established, optimized and validated. In addition, it was projected to validate a molecular system for differentiation of bread wheat and spelt; however, attempts for molecular differentiation between common wheat and spelt based on the gene GAG56D failed because of the genetic variability of the molecular target. Primer–probe systems were further developed and optimized on the basis of alignments of DNA sequences, as well as already developed PCR systems. The specificity of each system was demonstrated on 10 (spelt), 11 (durum wheat and rye) and 12 (bread wheat) reference samples. Specificity of the barley system was already proved in previous work. The calculated limits of detection (LOD95%) were between 2.43 and 4.07 single genome copies in real-time PCR. Based on the “three droplet rule”, the LOD95% in ddPCR was calculated to be 9.07–13.26 single genome copies. The systems were tested in mixtures of flours (rye and common wheat) and of semolina (durum and common wheat). The methods proved to be robust with regard to the tested conditions in the ddPCR. The developed primer–probe systems for ddPCR proved to be effective in quantitatively detecting the investigated cereal species rye and common wheat in mixtures by taking into account the haploid genome weight and the degree of milling of a flour. This method can correctly detect proportions of 50%, 60% and 90% wholemeal rye flour in a mixture of wholemeal common wheat flour. Quantitative results depend on the DNA content, on ploidy of cereal species and are also influenced by comminution. Hence, the proportion of less processed rye is overestimated in higher processed bread wheat and adulteration of durum wheat by common wheat by 1–5% resulted in underestimation of common wheat.

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A Multiplex, Droplet Digital PCR Assay for the Detection of T-Cell Receptor Excision Circles and Kappa-Deleting Recombination Excision Circles

Clinical Chemistry ◽

10.1373/clinchem.2019.308171 ◽

2019 ◽

Vol 66 (1) ◽

pp. 229-238 ◽

Cited By ~ 1

Author(s):

Tracie Profaizer ◽

Patricia Slev

Keyword(s):

Flow Cytometry ◽

T Cell ◽

T Cell Receptor ◽

Reference Gene ◽

Cell Receptor ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Blood Samples ◽

Healthy Donors ◽

Excision Circles

Abstract BACKGROUND T-cell receptor excision circles (TREC) and κ-deleting recombination receptor excision circles (KREC) concentrations can be used to assess and diagnose immune deficiencies, monitor thymic and bone marrow immune reconstitution, or follow responses to drug therapy. We developed an assay to quantify TREC, KREC, and a reference gene in a single reaction using droplet digital PCR (ddPCR). METHODS PCR was optimized for 3 targets: TREC, KREC, and ribonuclease P/MRP subunit p30 (RPP30) as the reference gene. Multiplexing was accomplished by varying the target's fluorophore and concentration. Correlation with clinical results was evaluated using 47 samples from healthy donors, 59 samples with T-cell and B-cell markers within the reference interval from the flow cytometry laboratory, 20 cord blood samples, and 34 samples submitted for exome sequencing for severe combined immunodeficiency disease (SCID). RESULTS The limit of the blank was 4 positive droplets, limit of detection 9 positive droplets, and limit of quantification 25 positive droplets, or 2.0 copies/μL. TREC and KREC copies/μL were as expected in the healthy donors and cord blood samples and concordant with the healthy flow cytometry results. Of the samples from the SCID Panel, 56.5% had a TREC count <20 copies/μL and 17.7% had a KREC count <20 copies/μL, suggestive of low T- and B-cell numbers, respectively. CONCLUSIONS Our multiplex ddPCR assay is an analytically sensitive and specific method for the absolute quantification of TREC and KREC. To the best of our knowledge, this paper is the first to describe the simultaneous quantification of TREC, KREC, and a reference gene by use of ddPCR.

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Detection of transgenic rice line TT51-1 in processed foods using conventional PCR, real-time PCR, and droplet digital PCR

Food Control ◽

10.1016/j.foodcont.2018.11.032 ◽

2019 ◽

Vol 98 ◽

pp. 380-388 ◽

Cited By ~ 3

Author(s):

Xiaofu Wang ◽

Ting Tang ◽

Qingmei Miao ◽

Shilong Xie ◽

Xiaoyun Chen ◽

...

Keyword(s):

Real Time ◽

Transgenic Rice ◽

Real Time Pcr ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Processed Foods ◽

Conventional Pcr ◽

Rice Line ◽

Transgenic Rice Line

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Comprehensive Evaluation of the Effects of Long-term Cryopreservation on Peripheral Blood Mononuclear Cells using Flow Cytometry

10.21203/rs.3.rs-1059931/v1 ◽

2021 ◽

Author(s):

Bo Li ◽

Chunmei Yang ◽

Gui Ja ◽

Yansheng Liu ◽

Na Wang ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Peripheral Blood Mononuclear Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Peripheral Blood Mononuclear ◽

Effective Utilization ◽

Blood Mononuclear Cells ◽

Important Evidence

Abstract Human peripheral blood mononuclear cells (PBMCs) originate from hematopoietic stem cells (HSCs) in the bone marrow, which mainly includes lymphocytes (T cells, B cells, and natural killer [NK] 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.

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