Serial Measurements of Apoptotic Cell Numbers Provide  Better Acceptance Criterion for PBMC Quality than a  Single Measurement Prior to the T Cell Assay

Wunsch, Marie; Caspell, Richard; Küerten, Stefanie; Lehmann, Paul; Sundararaman, Srividya

doi:10.3390/cells4010040

Cited by 12 publications

(13 citation statements)

References 36 publications

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“…1B), suggesting that the majority of the cells, regardless of their viability, were analyzed, without significant cell loss during the freezing process. Consistent with what was previously reported (22,28), cryopreservation affected PBMC viability in all of the groups studied ( Fig. 2A).…”

Section: Discussionsupporting

confidence: 80%

“…To establish whether natural DV infection in children affects the viability of cryopreserved PBMCs, the frequencies of nonviable PBMCs before and after freezing were evaluated. As previously reported (22), increased frequencies of nonviable cells were found in PBMCs after cryopreservation in all of the groups analyzed (P Յ 0.0001, Wilcoxon test) ( Fig. 2A), confirming the effect of the process on cellular viability.…”

Section: Resultssupporting

confidence: 73%

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Viability and Functionality of Cryopreserved Peripheral Blood Mononuclear Cells in Pediatric Dengue

Perdomo‐Celis

Salgado

Castañeda

et al. 2016

Clin Vaccine Immunol

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bCryopreserved peripheral blood mononuclear cells (PBMCs) are widely used in studies of dengue. In this disease, elevated frequency of apoptotic PBMCs has been described, and molecules such as soluble tumor necrosis factor (TNF)-related apoptosisinducing ligands (sTRAIL) are involved. This effect of dengue may affect the efficiency of PBMC cryopreservation. Here, we evaluate the viability (trypan blue dye exclusion and amine-reactive dye staining) and functionality (frequency of gamma interferon [IFN-␥]-producing T cells after polyclonal stimulation) of fresh and cryopreserved PBMCs from children with dengue (in acute and convalescence phases), children with other febrile illnesses, and healthy children as controls. Plasma sTRAIL levels were also evaluated. The frequencies of nonviable PBMCs detected by the two viability assays were positively correlated (r ‫؍‬ 0.74; P < 0.0001). Cryopreservation particularly affected the PBMCs of children with dengue, who had a higher frequency of nonviable cells than healthy children and children with other febrile illnesses (P < 0.02), and PBMC viability levels were restored in the convalescent phase. In the acute phase, an increased frequency of CD3 ؉ CD8 ؉ amine-positive cells was found before cryopreservation (P ‫؍‬ 0.01). Except for B cells in the acute phase, cryopreservation usually did not affect the relative frequencies of viable PBMC subpopulations. Dengue infection reduced the frequency of IFN-␥-producing CD3؉ cells after stimulation compared with healthy controls and convalescent-phase patients (P < 0.003), and plasma sTRAIL correlated with this decreased frequency in dengue (rho ‫؍‬ ؊0.56; P ‫؍‬ 0.01). Natural dengue infection in children can affect the viability and functionality of cryopreserved PBMCs. Cryopreservation is the maintenance of cells and biological tissues at low temperatures and is based on the use of various media or solutions that form hydrogen bonds with water molecules, preventing cellular damage. The low temperatures allow the cells to enter a quiescent state in which cellular functions are suspended without affecting their intrinsic characteristics (1). Peripheral blood mononuclear cells (PBMCs) are frequently cryopreserved for use in transplants or immunological studies (2, 3). However, the cryopreservation process may affect viability, phenotype, and cellular functionality due to factors such as inadequate temperatures, the freezing protocol used, the expertise of the personnel, and freezing time (4, 5). The disease of the individual from whom the PBMCs come also affects cryopreservation. For example, PBMCs from subjects infected with human immunodeficiency virus (HIV) presented reduced viability after cryopreservation, possibly due to the increased numbers of apoptotic cells circulating during the course of the disease (6). Similar findings have been found in the acute phases of diseases, such as visceral leishmaniasis (7). Particularly for HIV, great efforts have been undertaken to optimize the evaluation and comparability of immune tests...

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Section: Discussionsupporting

confidence: 80%

Section: Resultssupporting

confidence: 73%

Viability and Functionality of Cryopreserved Peripheral Blood Mononuclear Cells in Pediatric Dengue

Perdomo‐Celis

Salgado

Castañeda

et al. 2016

Clin Vaccine Immunol

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“…Antigens were plated first in CTL Test Medium TM (CTL, Cat #CTLT 005) in a volume of 100 μL per well at 2× the final concentrations. The plates containing the antigens were stored at 37 °C in a humidified CO 2 incubator until the PBMC were ready for plating [ 13 ]. The thawed PBMC were suspended in CTL Test Medium TM and added at 2.5 × 10 5 /cells per well in 100 μL—unless specified differently—using wide-bore pipette tips.…”

Section: Methodsmentioning

confidence: 99%

A Positive Control for Detection of Functional CD4 T Cells in PBMC: The CPI Pool

Schiller¹,

Zhang²,

Li³

et al. 2017

Cells

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Testing of peripheral blood mononuclear cells (PBMC) for immune monitoring purposes requires verification of their functionality. This is of particular concern when the PBMC have been shipped or stored for prolonged periods of time. While the CEF (Cytomegalo-, Epstein-Barr and Flu-virus) peptide pool has become the gold standard for testing CD8 cell functionality, a positive control for CD4 cells is so far lacking. The latter ideally consists of proteins so as to control for the functionality of the antigen processing and presentation compartments, as well. Aiming to generate a positive control for CD4 cells, we first selected 12 protein antigens from infectious/environmental organisms that are ubiquitous: Varicella, Influenza, Parainfluenza, Mumps, Cytomegalovirus, Streptococcus, Mycoplasma, Lactobacillus, Neisseria, Candida, Rubella, and Measles. Of these antigens, three were found to elicited interferon (IFN)-γ-producing CD4 cells in the majority of human test subjects: inactivated cytomegalo-, parainfluenza-, and influenza virions (CPI). While individually none of these three antigens triggered a recall response in all donors, the pool of the three (the ‘CPI pool’), did. One hundred percent of 245 human donors tested were found to be CPI positive, including Caucasians, Asians, and African-Americans. Therefore, the CPI pool appears to be suitable to serve as universal positive control for verifying the functionality of CD4 and of antigen presenting cells.

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“…However, after isolation of PBMC, early apoptotic events are present in both in vitro and ex vivo experiments; as a result, it is very important to determine apoptosis before using these cells in the experiments (Wunsch et al, 2015). Various methods have been used to measure apoptosis, including the YO-PRO-1/7-AAD method, which has been proposed as a good, low-cost alternative for sensitive detection of early apoptosis in PBMCs and >80% of viability is recommended before starting to work (Glisic-Milosavljevic et al, 2005).…”

Section: Technical Aspects To Consider Before Working With Pbmcs and mentioning

confidence: 99%

An Overview of Peripheral Blood Mononuclear Cells as a Model for Immunological Research of Toxoplasma gondii and Other Apicomplexan Parasites

Dávila

Rios

2019

Front. Cell. Infect. Microbiol.

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In biology, models are experimental systems meant to recreate aspects of diseases or human tissue with the goal of generating inferences and approximations that can contribute to the resolution of specific biological problems. Although there are many models for studying intracellular parasites, their data have produced critical contradictions, especially in immunological assays. Peripheral blood mononuclear cells (PBMCs) represent an attractive tissue source in pharmacogenomics and in molecular and immunologic studies, as these cells are easily collected from patients and can serve as sentinel tissue for monitoring physiological perturbations due to disease. However, these cells are a very sensitive model due to variables such as temperature, type of stimulus and time of collection as part of posterior processes. PBMCs have been used to study Toxoplasma gondii and other apicomplexan parasites. For instance, this model is frequently used in new therapies or vaccines that use peptides or recombinant proteins derived from the parasite. The immune response to T. gondii is highly variable, so it may be necessary to refine this cellular model. This mini review highlights the major approaches in which PBMCs are used as a model of study for T. gondii and other apicomplexan parasites. The variables related to this model have significant implications for data interpretation and conclusions related to host-parasite interaction.

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Serial Measurements of Apoptotic Cell Numbers Provide Better Acceptance Criterion for PBMC Quality than a Single Measurement Prior to the T Cell Assay

Cited by 12 publications

References 36 publications

Viability and Functionality of Cryopreserved Peripheral Blood Mononuclear Cells in Pediatric Dengue

Viability and Functionality of Cryopreserved Peripheral Blood Mononuclear Cells in Pediatric Dengue

A Positive Control for Detection of Functional CD4 T Cells in PBMC: The CPI Pool

An Overview of Peripheral Blood Mononuclear Cells as a Model for Immunological Research of Toxoplasma gondii and Other Apicomplexan Parasites

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