Database-guided Flow-cytometry for Evaluation of Bone Marrow Myeloid Cell Maturation

Aanei, Carmen Mariana; Jacob, Marie‐Christine; Veyrat-Masson, Richard; Picot, Tiphanie; Rosenthal-Allieri, Maria Alessandra; Lhoumeau, Anne-Catherine; Ticchioni, Michel; Dumézy, Florent; Catafal, Lydia Campos

doi:10.3791/57867

Cited by 4 publications

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“…After two successive washing steps with 2 ml phosphate-buffered saline (PBS), containing 0.2% bovine serum albumin (BSA), 0.009% sodium azide (AZ), and 0.07% ethylenediaminetetraacetic acid (EDTA) followed by centrifugation for 5 min at 400 × g, the samples were ready for acquisition on a FACSCanto II cytometer (BD Biosciences, San Jose, CA, USA) using BD FACSDiva v1.6 software. Details regarding sample preparation and the staining procedures were previously described ( 33 ).…”

Section: Methodsmentioning

confidence: 99%

Natural Killer Cell Subpopulations and Inhibitory Receptor Dynamics in Myelodysplastic Syndromes and Acute Myeloid Leukemia

et al. 2021

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Natural killer (NK) cells are key innate immunity effectors that play a major role in malignant cell destruction. Based on expression patterns of CD16, CD56, CD57, and CD94, three distinct NK cell maturation stages have been described, which differ in terms of cytokine secretion, tissue migration, and the ability to kill target cells. Our study addressed NK cell maturation in bone marrow under three conditions: a normal developmental environment, during pre-leukemic state (myelodysplastic syndrome, MDS), and during leukemic transformation (acute myeloblastic leukemia, AML). In this study, we used a new tool to perform multicolor flow cytometry data analysis, based on principal component analysis, which allowed the unsupervised, accurate discrimination of immature, mature, and hypermature NK subpopulations. An impaired NK/T cell distribution was observed in the MDS bone marrow microenvironment compared with the normal and AML settings, and a phenotypic shift from the mature to the immature state was observed in NK cells under both the MDS and AML conditions. Furthermore, an impaired NK cell antitumor response, resulting in changes in NK cell receptor expression (CD159a, CD158a, CD158b, and CD158e1), was observed under MDS and AML conditions compared with the normal condition. The results of this study provide evidence for the failure of this arm of the immune response during the pathogenesis of myeloid malignancies. NK cell subpopulations display a heterogeneous and discordant dynamic on the spectrum between normal and pathological conditions. MDS does not appear to be a simple, intermediate stage but rather serves as a decisive step for the mounting of an efficient or ineffective immune response, leading to either the removal of the tumor cells or to malignancy.

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Section: Methodsmentioning

confidence: 99%

Natural Killer Cell Subpopulations and Inhibitory Receptor Dynamics in Myelodysplastic Syndromes and Acute Myeloid Leukemia

et al. 2021

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“…Validate that the HL-60 cells have been successfully differentiated and are appropriate for use in the OPKA assay by testing viability and cell surface markers according to established flow cytometry protocols 16,17,18,19 . After differentiation, harvest HL-60 cells and stain approximately 1 × 10 4 cells with fluorescently-conjugated antibodies/stains for CD71, CD35, annexin V, and propidium iodide.…”

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confidence: 99%

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

Paschall

Middleton

Avci

2019

JoVE

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A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

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“…After two successive washing steps with 2 ml phosphatebuffered saline (PBS), containing 0.2% bovine serum albumin (BSA), 0.009% sodium azide (AZ), and 0.07% ethylenediaminetetraacetic acid (EDTA) followed by centrifugation for 5 min at 400 × g, the samples were ready for acquisition on a FACSCanto II cytometer (BD Biosciences, San Jose, CA, USA) using BD FACSDiva v1.6 software. Details regarding sample preparation and the staining procedures were previously described (33).…”

Section: Flow Cytometry Sample Preparationmentioning

confidence: 99%

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Database-guided Flow-cytometry for Evaluation of Bone Marrow Myeloid Cell Maturation

Cited by 4 publications

References 0 publications

Natural Killer Cell Subpopulations and Inhibitory Receptor Dynamics in Myelodysplastic Syndromes and Acute Myeloid Leukemia

Natural Killer Cell Subpopulations and Inhibitory Receptor Dynamics in Myelodysplastic Syndromes and Acute Myeloid Leukemia

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

DataSheet_1.pdf

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