Evaluating the Proliferation of Human PeripheralBlood Mononuclear Cells Using MTT Assay

Molaae, Nada; Mosayebi, Ghasem; Pishdadian, Abbas; Ejtehadifar, Mostafa; Ganji, Ali

doi:10.15171/ijbsm.2017.06

Cited by 33 publications

(16 citation statements)

References 13 publications

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“…Isolated PBMCs from 6 young and 4 aged beagles were incubated in either complete media or complete media containing ConA at a density of 1.5 × 10 6 cells/well, in a 48 well plate for 2 days. These mitogen stimulation conditions induce sub-maximal lymphoproliferative effects in prior canine and human studies, and are maintained for a shorter duration compared to previous studies utilizing ConA stimulated lymphocytes as a positive control (Boggiatto et al, 2010; Molaee et al, 2017; Wagner et al, 1999). Next, mitogen-stimulated and unstimulated control cells (1 × 10 6 cells) from each dog were stained for analysis by flow cytometry.…”

Section: Methodsmentioning

confidence: 96%

Multi-color flow cytometry for evaluating age-related changes in memory lymphocyte subsets in dogs

Withers

Moore

Chang

et al. 2018

Developmental & Comparative Immunology

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While dogs are increasingly being utilized as large-animal models of disease, important features of age-related immunosenescence in the dog have yet to be evaluated due to the lack of defined naïve vs. memory T lymphocyte phenotypes. We therefore performed multi-color flow cytometry on peripheral blood mononuclear cells from young and aged beagles, and determined the differential cytokine production by proposed memory subsets. CD4+ and CD8+ T lymphocytes in aged dogs displayed increased cytokine production, and decreased proliferative capacity. Antibodies targeting CD45RA and CD62L, but less so CD28 or CD44, defined canine cells that consistently exhibited properties of naïve-, central memory-, effector memory-, and terminal effector-like CD4+ and CD8+ T lymphocyte subsets. Older dogs demonstrated decreased frequencies of naïve-like CD4+ and CD8+ T lymphocytes, and an increased frequency of terminal effector-like CD8+ T lymphocytes. Overall findings revealed that aged dogs displayed features of immunosenescence similar to those reported in other species.

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Section: Methodsmentioning

confidence: 96%

Multi-color flow cytometry for evaluating age-related changes in memory lymphocyte subsets in dogs

Withers

Moore

Chang

et al. 2018

Developmental & Comparative Immunology

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“…Apparent anomalies in the MTS results were observed for PHA treatment, where although a statistically significant activation was observed for two out of three donors tested (p < 0.05-0.01), the recorded levels did not correspond to the level of cell proliferation observed on microscopic images for this condition (Fig 4). It is possible that the conditions used for PHA stimulation interfered with the results of the MTS assay such that the level of metabolic activity that this assay measured did not accurately correspond to the level of proliferation exhibited after PHA stimulation (28; 29).…”

Section: Resultsmentioning

confidence: 99%

Evidence for the Modulation of the Immune Response in Peripheral Blood Mononuclear Cells after Stimulation with a High Molecular Weight β-glucan Isolated fromLactobacillus fermentumLf2

Vitlić

Sadiq

et al. 2018

Preprint

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27Lactobacillus fermentum Lf 2 produces large amounts of exopolysaccharides under 28 optimized conditions (~2 g/L, EPS) which have been shown to possess 29 immunomodulatory activity. In this study, the crude EPS was fractionated to give a 30 high molecular weight (HMw) homoglycan and a mixture of medium molecular 31 weight heteroglycans. The HMw EPS was isolated and identified as a -glucan. 32 Peripheral blood mononuclear cells (PBMC) were pre-treated with purified 33 polysaccharide to determine if the HMw -glucan is responsible for the 34 immunomodulatory activity. Cells were also stimulated with either lipopolysaccharide 35 (LPS) or phytohemagglutinin (PHA) and their effects, both with and without -glucan 36 pre-treatment, compared. 37Exposure of the cells to -glucan increased their metabolic activity and whilst a small 38 but statistically significant drop in CD14 expression was observed at Day 1, the 39 levels were significantly elevated at Day 2. High levels of CD14 expression were 40 observed in cells initially exposed to the -glucan and subsequently stimulated with 41 either LPS or PHA. In contrast, reduced levels of TLR-2 expression were observed 42 for cells initially exposed to the -glucan and subsequently stimulated with LPS. 43TNF-α levels were elevated in -glucan treated cells (Day1) with the levels dropping 44 back once the -glucan had been removed (Day 2). The stimulants LPS and PHA 45 both induced significant rises in TNF- levels, however, this induction was 46 completely (LPS) or partially blocked (PHA) in -glucan pre-treated cells. 47 IMPORTANCE 50The use of foods containing probiotics (i.e. live microorganisms providing health 51 benefits) is growing in popularity. Whilst there is empirical evidence identifying 52 specific microorganisms as having beneficial effects, the precise mechanisms by 53 which these strains exert influence is not well understood. One of the problems is 54 that there are a large number of structures presented at the surface of 55 microorganisms which can interact with the host and which can initiate a host 56 response. In the current work, we addressed this issue by choosing a potential 57 probiotic strain of bacteria associated with cheese production, L. fermentum Lf2, 58 which produces large quantities of polysaccharides at its surface. From this 59 polysaccharide mixture, we have isolated, purified and characterised a HMw -60 glucan and demonstrated that it has immunomodulatory activity. This study 61 demonstrates that the -glucan produced by L. fermentum Lf2 can modify the 62 immune response of PBMC cells when subjected to a bacterial endotoxin. 63 64 4 1. Introduction 65 Species of Lactobacillus fermentum are obligate heterofermentative Gram-positive 66bacteria that can be isolated from a wide variety of sources and include members 67 that are normal inhabitants of the human microflora (1; 2). A significant number of 68 strains are closely associated with the production of fermented foods (3-7) and a 69 number have been exploited for their probioti...

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“…(19,20) In several studies, it was explained that IDO inhibits airway proinflammation mediated by Th2 cells, but does not seem to influence tolerance to inhibition in the immune response of proinflammatory cells Th1. (20,21,22) Conclusion Cellular immune profile of subjects with allergic asthma to Dermatophagoides pterronyssinus (Der p) is characterized by a type-2 inflammatory response that is dominant compared to type-1 inflammation (higher IL-13 ratio compared to IFN-γ) and to the role of anti-inflammation (higher IL-13 ratio compared to IL-10). The decline in IDO production in allergic asthma subjects to Der p is thought to be related to the low cellular immune response in expressing IFN-γ compared to IL-13.…”

Section: Discussionmentioning

confidence: 99%

Interleukin-13, Interleukin-10, Interferon-gamma and IDO Production in Response to Home Dust Mites in Allergic Asthma

et al. 2019

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BACKGROUND: Allergic asthma is a degenerative atopic disease caused by allergic or hypersensitivity type-1. More than 50% of people with allergic asthma are caused by the presence of house dust mites (HDMs) allergens.METHODS: The cellular immunity response was evaluated through a peripheral blood mononuclear cell (PBMC) culture isolated from blood, using the ficoll gradient technique. Subjects were atopic asthma groups and non-atopic asthma groups. PBMC from each subject cultured was stimulated with HDMs allergen, then incubated in a CO2 5% incubator, 37o C for 72 hours. With the multiplex assay method, interferon (IFN)-γ, interleukin (IL)-13 and IL-10 were measured, meanwhile indoleamine 2,3-dioxygenase level (IDO) was measured by the enzyme-linked immunosorbent assay (ELISA) sandwich methods.RESULTS: The IFN-γ production in the supernatant of PBMC cultures was stimulated by phytohemagglutinin (PHA), Roswell Park Memorial Institute (RPMI) medium and allergens. The IFN-γ production in allergen-stimulated supernatants showed higher level of IFN-γ in the nonatopic group (4,681,455±3,434,851) than atopic group (4,363,300±2,067,941) even though it was not statistically significant (p=0.078). There were no differences between the mean of IL-13 production in atopic asthma group and non-atopic group. The IL-10 production in allergenstimulated supernatants was shown to be higher in nonatopic group and were statistically significantly different (p=0.015). The IDO production in allergen-stimulated supernatants was shown to be higher in the non-atopic group (272,231±269,564) than in the actopic group (13,273±400), and it was significantly different (p=0.007).CONCLUSION: Cellular immune profile of subjects with allergic asthma to Dermatophagoides pterronyssinus (Der p) is characterized by a type-2 inflammatory response that is dominant compared to type-1 inflammation (higher IL-13 ratio compared to IFN-γ) and to the role of anti-inflammation (higher IL-13 ratio compared to IL-10). The decline in IDO production in allergic asthma subjects to Der p is thought to be related to the low cellular immune response in expressing IFN-γ compared to IL-13.KEYWORDS: interleukin-13, interleukin-10, IDO, PBMC, asthma

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Evaluating the Proliferation of Human PeripheralBlood Mononuclear Cells Using MTT Assay

Cited by 33 publications

References 13 publications

Multi-color flow cytometry for evaluating age-related changes in memory lymphocyte subsets in dogs

Multi-color flow cytometry for evaluating age-related changes in memory lymphocyte subsets in dogs

Evidence for the Modulation of the Immune Response in Peripheral Blood Mononuclear Cells after Stimulation with a High Molecular Weight β-glucan Isolated fromLactobacillus fermentumLf2

Interleukin-13, Interleukin-10, Interferon-gamma and IDO Production in Response to Home Dust Mites in Allergic Asthma

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