Application of JC1 for non-toxic isolation of cells with MDR transporter activity by flow cytometry

Wolosin, J. Mario; Zamudio, Aldo C.; Wang, Zheng

doi:10.1371/journal.pone.0174905

Cited by 11 publications

(8 citation statements)

References 32 publications

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“…PI uptake vs. exclusion was used to discriminate dead necrotic cells with permeable plasma membranes (PIpositive) from live cells with intact membranes (PI-negative). For the analysis of cell apoptosis, the mitochondrial membrane potential (MMP) probe JC-1 (5,5 ′ ,6,6 ′ -tetrachloro-1,1 ′ ,3,3 ′tetraethylbenzimidazolcarbocyanine iodide) was used as previously described (35,36). Separated camel leukocytes (100 µL) were plated in a 96-well microtiter plate at a density of 5 × 10 6 cell/mL in RPMI cell culture medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, and 10 mg/mL streptomycin (Gibco Laboratories, Carlsbad, CA).…”

Section: Leukocyte Viability Assaymentioning

confidence: 99%

Changes in Cell Vitality, Phenotype, and Function of Dromedary Camel Leukocytes After Whole Blood Exposure to Heat Stress in vitro

Hussen

2021

Front. Vet. Sci.

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The dromedary camel (Camelus dromedarius) is well-adapted to the desert environment with the ability to tolerate increased internal body temperatures rising daily to 41–42°C during extreme hot. This study was undertaken to assess whether in vitro incubation of camel blood at 41°C, simulating conditions of heat stress, differently alters cell vitality, phenotype, and function of leukocytes, compared to incubation at 37°C (normothermia). Using flow cytometry, the cell vitality (necrosis and apoptosis), the expression of several cell markers and adhesion molecules, and the antimicrobial functions of camel leukocytes were analyzed in vitro. The fraction of apoptotic cells within the granulocytes, lymphocytes, and monocytes increased significantly after incubation of camel whole blood at 41°C for 4 h. The higher increase in apoptotic granulocytes and monocytes compared to lymphocytes suggests higher resistance of camel lymphocytes to heat stress. Functionally, incubation of camel blood at 41°C for 4 h enhanced the phagocytosis and ROS production activities of camel neutrophils and monocytes toward S. aureus. Monocytes from camel blood incubated at 41°C for 4 h significantly decreased their expression level of MHC class II molecules with no change in the abundance of CD163, resulting in a CD163high MHC-IIlow M2-like macrophage phenotype. In addition, heat stress treatment showed an inhibitory effect on the LPS-induced changes in camel monocytes phenotype. Furthermore, in vitro incubation of camel blood at 41°C reduced the expression of the cell adhesion molecules CD18 and CD11a on neutrophils and monocytes. Collectively, the present study identified some heat-stress-induced phenotypic and functional alterations in camel blood leukocytes, providing a paradigm for comparative immunology in the large animals. The clinical relevance of the observed changes in camel leukocytes for the adaptation of the camel immune response to heat stress conditions needs further in vitro and in vivo studies.

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Section: Leukocyte Viability Assaymentioning

confidence: 99%

Changes in Cell Vitality, Phenotype, and Function of Dromedary Camel Leukocytes After Whole Blood Exposure to Heat Stress in vitro

Hussen

2021

Front. Vet. Sci.

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“…Cell apoptosis was analyzed using the mitochondrial membrane potential (MMP) probe JC-1 [23,24]. Leukocytes (6× 10 5 cell in 100 µL RPMI cell culture medium) were plated in a microtiter plate (96-well).…”

Section: Analysis Of Cell Necrosis and Apoptosismentioning

confidence: 99%

The Impact of Anticoagulation Agent on the Composition and Phenotype of Blood Leukocytes in Dromedary Camels

Hussen

Shawaf

Alhojaily

2022

Veterinary Sciences

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For the analysis of several cellular biomarkers, blood samples are anticoagulated using different agents with different modes of action. However, for the most commonly used anticoagulants, EDTA and heparin, varying effects on blood components have been reported in different species. As little is known about the impact of anticoagulants on the immunological evaluation of camel leukocytes, the present study analyzed the leukogram, the immunophenotype, and the cell vitality of camel leukocytes separated from blood samples anticoagulated with EDTA or lithium heparin. Using flow cytometry and staining with monoclonal antibodies to several cell surface markers, the composition and immunophenotype of camel leukocytes separated from blood anticoagulated with EDTA or heparin were analyzed. In comparison to EDTA-anticoagulated blood, using lithium heparin as an anticoagulant resulted in reduced numbers of total leukocytes and reduced numbers of neutrophils, which led to a reduced neutrophil to lymphocyte ratio. The analysis of cell necrosis and apoptosis after the staining of leukocytes with the DNA-sensitive dye propidium iodide and the mitochondrial membrane potential probe JC1 revealed a higher fraction of necrotic neutrophils and higher fractions of apoptotic neutrophils and monocytes in heparin blood than in EDTA blood. In addition, monocytes from heparin blood showed higher expression levels of the cell surface markers CD14, CD163, and MHCII when compared to cells from EDTA blood. Similarly, in heparin blood, CD44 and CD172a were expressed higher on neutrophils, while CD11a was expressed higher on lymphocytes in comparison to cells from EDTA blood. The results of the current study indicate the importance of considering the type of anticoagulant when investigating the composition, vitality, and immunophenotype of camel leukocytes.

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“…To confirm the association between gene mutations and ABC proteins, we explored the expression levels of ABC genes known to be associated with prognosis in AML and whose functionality is putatively tested with JC-1 +/-CsA assay 19,[28][29][30] . We explored ABC gene expression according to gene mutational status in a previously published series of 405 AML patients 31 .…”

Section: Abc Activity Discriminates De Novo-type Aml From Other Ontog...mentioning

confidence: 99%

Clinical and biological impact of ATP-binding cassette transporter activity in adult acute myeloid leukemia

et al. 2022

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Chemotherapy resistance is the main cause of treatment failure in acute myeloid leukemia (AML) and has been related to ATP-binding cassette (ABC) transporter activity. However, the links between ABC activity, immunophenotype, and molecular AML parameters have been poorly evaluated. Moreover, the prognostic value of ABC activity, when compared to new molecular markers, is unknown. Here we investigated the links between ABC activity, as evaluated by JC-1 +/- cyclosporine A (CsA) assay, and immunophenotypic, cytogenetic, molecular, and targeted next generation sequencing features in 361 AML patients. High ABC activity was found in 164 patients and was significantly associated with less proliferating disease, an immature immunophenotype (expression of CD34, HLA-DR, CD117, CD13), and gene mutations defining AML as belonging to secondary-type ontogenic groups. Low ABC activity was associated with more mature myeloid differentiation (CD34-, cyMPO+, CD15+, CD33+) or monocytic commitment (CD64+, CD4+weak, CD14+), with NPM1 mutations, KMT2A rearrangements, and core-binding factor gene fusions, hallmarks of the de novotype AML ontogeny. ABC activity was one of the major factors we identified using a random forest model for early prediction of AML ontogeny. In the 230 patients evaluated at diagnosis and intensively treated, high ABC activity was a predictive factor for primary resistance, and in multivariate analysis including full molecular data, an independent factor for event-freesurvival (p=0.0370). JC-1 +/- CsA assay could be used at diagnosis to predict AML ontogeny and to complete prognosis evaluation in addition to new molecular markers.

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Application of JC1 for non-toxic isolation of cells with MDR transporter activity by flow cytometry

Cited by 11 publications

References 32 publications

Changes in Cell Vitality, Phenotype, and Function of Dromedary Camel Leukocytes After Whole Blood Exposure to Heat Stress in vitro

Changes in Cell Vitality, Phenotype, and Function of Dromedary Camel Leukocytes After Whole Blood Exposure to Heat Stress in vitro

The Impact of Anticoagulation Agent on the Composition and Phenotype of Blood Leukocytes in Dromedary Camels

Clinical and biological impact of ATP-binding cassette transporter activity in adult acute myeloid leukemia

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