Evaluation of EBV transformation of human memory B-cells isolated by FACS and MACS techniques

Sadreddini, Sanam; Jadidi‐Niaragh, Farhad; Younesi, Vahid; Pourlak, Tala; Afkham, Amir; Shokri, Fazel; Yousefi, Mehdi

doi:10.3109/1547691x.2015.1132288

Cited by 6 publications

(7 citation statements)

References 24 publications

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“…Few rigorous attempts to characterize and measure the effects of cell sorting on a range of sample types using modern technologies have been undertaken. [2][3][4][5][6][7] However, an understanding of the effects of cell sorting on samples of various kinds can guide researchers to predict whether sorting could influence a critical result and inform how to plan for addition controls or validation. In this study, cell cycle analysis and gene expression results from 3 sorting experiments using different cell types that encompassed different sites, instrument models, nozzle diameters, pressures, and exposure to UV laser illumination were performed.…”

Section: Discussionmentioning

confidence: 99%

“…2 In another study, Richardson et al 3 observed minimal gene expression changes in different populations of mouse mammary cell subsets when examined immediately after cell sorting. Sadreddini et al 4 demonstrated that magnetically activated cell sorting resulted in a higher transformation efficiency of memory B cells by Epstein-Barr virus when compared with traditional cell sorting. A study that examined the effect of sorting on human peripheral blood CD8 + cells observed sorting-induced activation of MAPK p38 with minimal functional changes.…”

Section: Introductionmentioning

confidence: 99%

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Evaluating the Effects of Cell Sorting on Gene Expression

et al. 2020

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Cell sorting is a commonly used technology to isolate highly purified cell populations for downstream applications. Because the sorted cells are destined for further analysis, i.e., gene expression assays or functional assays, ensuring that the sorting process itself has little effect on the cells is of utmost importance. Previous studies examining the effects of sorting on cellular function have primarily focused on a specific cell type or condition. One of the goals of the Flow Cytometry Research Group of the Association of Biomolecular Resource Facilities is to establish best practice guidelines for cell sorting conditions that minimize cell stress, perturbation, or injury to the sorted cell population. In this study, the effects of nozzle size, sample pressure, UV exposure, and instrument type were evaluated for their effects on gene expression and cell cycle using both established cell lines and primary cells across several flow cytometry shared facilities. Results indicate that nozzle size and pressure, as well as UV exposure and instrument type, have only minor effects on gene expression, which were diminished by subsequent culturing of the sorted cells. In this assessment, these data demonstrate that cell sorting itself, regardless of instrumentation used, has minimal effects on downstream cellular applications.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Evaluating the Effects of Cell Sorting on Gene Expression

et al. 2020

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“…The sorted cells can be used in a variety of applications including disease modeling, drug testing and characterization (i.e., lineage and gene expression) samples. For example, Sadreddini et al 45 investigated the effect of cell sorting on the transformation efficiency of antibody producing memory B-cells. The B-cells had been transformed with Epstein-Barr virus which caused them to transform into immortal lymphoblastoid cells and proliferate indefinitely.…”

Section: Isolation Of Naturally Occurring Cell Subpopulationsmentioning

confidence: 99%

Applications of flow cytometry sorting in the pharmaceutical industry: A review

Vitelli

Budman

Pritzker

et al. 2021

Biotechnol Progress

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The article reviews applications of flow cytometry sorting in manufacturing of pharmaceuticals. Flow cytometry sorting is an extremely powerful tool for monitoring, screening and separating single cells based on any property that can be measured by flow cytometry. Different applications of flow cytometry sorting are classified into groups and discussed in separate sections as follows: (a) isolation of cell types, (b) high throughput screening, (c) cell surface display, (d) droplet fluorescent-activated cell sorting (FACS). Future opportunities are identified including: (a) sorting of particular fractions of the cell population based on a property of interest for generating inoculum that will result in improved outcomes of cell cultures and (b) the use of population balance models in combination with FACS to design and optimize cell cultures.

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“…Thus, the exposure of cells within the sorter to factors that may alter their native state has the potential to affect the function of the sorted cell population(s) (ie, cytokine production, proliferation) and ultimately affect experimental results, especially when sorting to single cells. [1], [2], [3], [4], [5], [6], [7], [8] In any laboratory, experiments must be performed in controlled, clean environments to minimize the alteration of results because of external factors. Common laboratory biological contaminants include bacteria, molds, yeasts, and mycoplasma.…”

Section: Introductionmentioning

confidence: 99%

Cell Sorter Cleaning Practices and Their Impact on Instrument Sterility

Box¹,

Holmes²,

DeLay³

et al. 2022

J Biomol Tech

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Cells isolated using electrostatic cell sorters are subsequently evaluated in a variety of in vitro and in vivo applications. Thus, manipulations to the cells during the pre-and post-sort processing as well as when the cells are being analyzed by and passing through the sorter fluidics has the potential to affect the experimental results. There are many variables to consider when seeking to preserve cellular integrity and function during the cell-sorting process. A previous study by the Association of Biomolecular Resource Facilities Flow Cytometry Research Group (FCRG) investigated downstream effects on different cell types as a function of sorting variables such as pressure, nozzle size, and temperature. This multisite study revealed site-to-site variability based on differential gene expression when the same cell type and sort conditions were used. These results indicated the possibility that environmental factors such as the presence of contaminants in the sorter fluidics could exhibit effects on downstream molecular assays (ie, endotoxins or RNases). In the study described here, the FCRG sought to better understand how sorters are maintained and evaluated for contaminants such as bacteria, endotoxin, and RNases. In addition, the efficacy of an endotoxin decontamination method was evaluated. The results demonstrated that the majority of sorters in shared resource laboratories are free of RNase activity and bacteria; however, many are contaminated with endotoxin. The efficacy of a hydrogen peroxide cleaning procedure was tested and found to exhibit only a short-term effectiveness in eliminating endotoxin contamination.

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Evaluation of EBV transformation of human memory B-cells isolated by FACS and MACS techniques

Cited by 6 publications

References 24 publications

Evaluating the Effects of Cell Sorting on Gene Expression

Evaluating the Effects of Cell Sorting on Gene Expression

Applications of flow cytometry sorting in the pharmaceutical industry: A review

Cell Sorter Cleaning Practices and Their Impact on Instrument Sterility

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