Immunomagnetic separation is a suitable method for electrophysiology and ion channel pharmacology studies on T cells

Tajti, Gábor; Szanto, Tibor G.; Csóti, Agota; Racz, Greta; Evaristo, César; Hajdú, Péter; Panyi, György

doi:10.1080/19336950.2020.1859753

Cited by 6 publications

(3 citation statements)

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“…As a result, the free binding sites can be used to determine T cells via the CD3 molecule [ 33 ]. In line with these data, T-helper cells isolated by CD4-REAlease ® were reported to be suitable for electrophysiology and ion channel studies [ 34 ] and we showed previously that CD14-TACS ® separation of monocytes yielded intact cells that can be used to generate functional dendritic cells [ 4 ].…”

Section: Discussionsupporting

confidence: 56%

Comparison of Three CD3-Specific Separation Methods Leading to Labeled and Label-Free T Cells

Weiß

Gerdes²,

Berthold³

et al. 2021

Cells

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T cells are an essential part of the immune system. They determine the specificity of the immune response to foreign substances and, thus, help to protect the body from infections and cancer. Recently, T cells have gained much attention as promising tools in adoptive T cell transfer for cancer treatment. However, it is crucial not only for medical purposes but also for research to obtain T cells in large quantities, of high purity and functionality. To fulfill these criteria, efficient and robust isolation methods are needed. We used three different isolation methods to separate CD3-specific T cells from leukocyte concentrates (buffy coats) and Ficoll purified PBMCs. To catch the target cells, the Traceless Affinity Cell Selection (TACS®) method, based on immune affinity chromatography, uses CD-specific low affinity Fab-fragments; while the classical Magnetic Activated Cell Sorting (MACS®) method relies on magnetic beads coated with specific high affinity monoclonal antibodies. The REAlease® system also works with magnetic beads but, in contrast to MACS®, low-affinity antibody fragments are used. The target cells separated by TACS® and REAlease® are “label-free”, while cells isolated by MACS® still carry the cell specific label. The time required to isolate T cells from buffy coat by TACS® and MACS® amounted to 90 min and 50 min, respectively, while it took 150 min to isolate T cells from PBMCs by TACS® and 110 min by REAlease®. All methods used are well suited to obtain T cells in large quantities of high viability (>92%) and purity (>98%). Only the median CD4:CD8 ratio of approximately 6.8 after REAlease® separation differed greatly from the physiological conditions. MACS® separation was found to induce proliferation and cytokine secretion. However, independent of the isolation methods used, stimulation of T cells by anti CD3/CD28 resulted in similar rates of proliferation and cytokine production, verifying the functional activity of the isolated cells.

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Section: Discussionsupporting

confidence: 56%

Comparison of Three CD3-Specific Separation Methods Leading to Labeled and Label-Free T Cells

Weiß

Gerdes²,

Berthold³

et al. 2021

Cells

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“…The IMS technology has been demonstrated to have the following qualities: high sensitivity, excellent specificity and a rapid separation speed [178]. Among the disadvantages of IMS approaches is the difficulty of isolating complex phenotypes [179]. Immunomagnetic separation and fluorescence-activated cell sorting both have known or unknown drawbacks.…”

Section: Immunomagnetic Separation Technology (Ims)mentioning

confidence: 99%

An Overview of the Public Health Challenges in Diagnosing and Controlling Human Foodborne Pathogens

et al. 2023

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Pathogens found in food are believed to be the leading cause of foodborne illnesses; and they are considered a serious problem with global ramifications. During the last few decades, a lot of attention has been paid to determining the microorganisms that cause foodborne illnesses and developing new methods to identify them. Foodborne pathogen identification technologies have evolved rapidly over the last few decades, with the newer technologies focusing on immunoassays, genome-wide approaches, biosensors, and mass spectrometry as the primary methods of identification. Bacteriophages (phages), probiotics and prebiotics were known to have the ability to combat bacterial diseases since the turn of the 20th century. A primary focus of phage use was the development of medical therapies; however, its use quickly expanded to other applications in biotechnology and industry. A similar argument can be made with regards to the food safety industry, as diseases directly endanger the health of customers. Recently, a lot of attention has been paid to bacteriophages, probiotics and prebiotics most likely due to the exhaustion of traditional antibiotics. Reviewing a variety of current quick identification techniques is the purpose of this study. Using these techniques, we are able to quickly identify foodborne pathogenic bacteria, which forms the basis for future research advances. A review of recent studies on the use of phages, probiotics and prebiotics as a means of combating significant foodborne diseases is also presented. Furthermore, we discussed the advantages of using phages as well as the challenges they face, especially given their prevalent application in food safety.

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“…The hKv1.2 current was recorded in CHO cells, which heterologously expressed the hKv1.2 ion channel, and hKv1.3 current was recorded in the human peripheral blood lymphocytes. The stimulation of the hKv1.3 channel expression (activation of isolated mononuclear cells by PHA, see the Materials and Methods section) and recording conditions (no Ca 2+ in the pipette to elicit Ca 2+ -activated K + channels) guaranteed that the current recorded in these cells is K + current through Kv1.3 ( Varga et al, 2012 ; Bartok et al, 2014 ; Tajti et al, 2021 ). A custom-built perfusion system was used to apply toxins at a very small perfusion rate of 200 µl/min.…”

Section: Resultsmentioning

confidence: 99%

Optimization of Pichia pastoris Expression System for High-Level Production of Margatoxin

et al. 2021

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Margatoxin (MgTx) is a high-affinity blocker of voltage-gated potassium (Kv) channels. It inhibits Kv1.1–Kv1.3 ion channels in picomolar concentrations. This toxin is widely used to study physiological function of Kv ion channels in various cell types, including immune cells. Isolation of native MgTx in large quantities from scorpion venom is not affordable. Chemical synthesis and recombinant production in Escherichia coli need in vitro oxidative refolding for proper disulfide bond formation, resulting in a very low yield of peptide production. The Pichia pastoris expression system offers an economical approach to overcome all these limitations and gives a higher yield of correctly refolded recombinant peptides. In this study, improved heterologous expression of recombinant MgTx (rMgTx) in P. pastoris was obtained by using preferential codons, selecting the hyper-resistant clone against Zeocin, and optimizing the culturing conditions. About 36 ± 4 mg/L of >98% pure His-tagged rMgTx (TrMgTx) was produced, which is a threefold higher yield than has been previously reported. Proteolytic digestion of TrMgTx with factor Xa generated untagged rMgTx (UrMgTx). Both TrMgTx and UrMgTx blocked the Kv1.2 and Kv1.3 currents (patch-clamp) (Kd for Kv1.2 were 64 and 14 pM, and for Kv1.3, 86 and 50 pM, respectively) with comparable potency to the native MgTx. The analysis of the binding kinetics showed that TrMgTx had a lower association rate than UrMgTx for both Kv1.2 and Kv1.3. The dissociation rate of both the analogues was the same for Kv1.3. However, in the case of Kv1.2, TrMgTx showed a much higher dissociation rate with full recovery of the block than UrMgTx. Moreover, in a biological functional assay, both peptides significantly downregulated the expression of early activation markers IL2R and CD40L in activated CD4+ TEM lymphocytes whose activation was Kv1.3 dependent. In conclusion, the authors report that the Pichia expression system is a powerful method to produce disulfide-rich peptides, the overexpression of which could be enhanced noticeably through optimization strategies, making it more cost-effective. Since the presence of the His-tag on rMgTx only mildly altered the block equilibrium and binding kinetics, recombinant toxins could be used in ion channel research without removing the tag and could thus reduce the cost and time demand for toxin production.

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Immunomagnetic separation is a suitable method for electrophysiology and ion channel pharmacology studies on T cells

Cited by 6 publications

References 50 publications

Comparison of Three CD3-Specific Separation Methods Leading to Labeled and Label-Free T Cells

Comparison of Three CD3-Specific Separation Methods Leading to Labeled and Label-Free T Cells

An Overview of the Public Health Challenges in Diagnosing and Controlling Human Foodborne Pathogens

Optimization of Pichia pastoris Expression System for High-Level Production of Margatoxin

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