Single-colour flow cytometric assay to determine NK cell-mediated cytotoxicity and viability against non-adherent human tumor cells

Thakur, Ajit; Zaman, Abeyat; Hummel, Jeff; Jones, Kim S.; Hortelano, Gonzalo

doi:10.1007/s10529-011-0828-9

Cited by 6 publications

(3 citation statements)

References 15 publications

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“…To assess the bioactivity of Tandab (CD3/CD19) secreted by MSCs, a co-culture system using transwell plates with 0.4-μm-pore membrane was established. The specific lysis of target cells was determined by FACS analysis according to the “calcein-loss” method [ 26 ]. Briefly, MSC-Tandab, MSC-EV, or MSCs were seeded into 6-well culture plates at a density of 1 × 10 5 cells per well and incubated for 72 h. Then, Raji cells labeled with calcein-AM (Sigma, USA) and pre-activated PBMCs were added to the equilibrated inserts at an E:T ratio of 10:1.…”

Section: Methodsmentioning

confidence: 99%

Mesenchymal stromal cells as vehicles of tetravalent bispecific Tandab (CD3/CD19) for the treatment of B cell lymphoma combined with IDO pathway inhibitor d-1-methyl-tryptophan

et al. 2017

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BackgroundAlthough blinatumomab, a bispecific T cell engaging antibody, exhibits high clinical response rates in patients with relapsed or refractory B-precursor acute lymphoblastic leukemia (B-ALL) and B cell non-Hodgkin’s lymphoma (B-NHL), it still has some limitations because of its short half-life. Mesenchymal stromal cells (MSCs) represent an attractive approach for delivery of therapeutic agents to cancer sites owing to their tropism towards tumors, but their immunosuppression capabilities, especially induced by indoleamine 2,3-dioxygenase (IDO), should also be taken into consideration.MethodsHuman umbilical cord-derived MSCs (UC-MSCs) were genetically modified to secrete Tandab (CD3/CD19), a tetravalent bispecific tandem diabody with two binding sites for CD3 and two for CD19. The tropism of MSCs towards Raji cells in vitro was determined by migration assays, and the homing property of MSCs in vivo was analyzed with firefly luciferase-labeled MSCs (MSC-Luc) by bioluminescent imaging (BLI). The cytotoxicity of T cells induced by MSC-secreting Tandab (CD3/CD19) was detected in vitro and in vivo in combination with d-1-methyl-tryptophan (D-1MT), an IDO pathway inhibitor.ResultsThe purified Tandab (CD3/CD19) was functional with high-binding capability both for CD3-positive cells and CD19-positive cells and was able to induce specific lysis of CD19-positive cell lines (Raji, Daudi, and BJAB) in the presence of T cells. Additionally, results from co-culture killing experiments demonstrated that Tandab (CD3/CD19) secreted from MSCs was also effective. Then, we confirmed that D-1MT could enhance the cytotoxicity of T cells triggered by MSC-Tandab through reversing T cell anergy with down-regulation of CD98 and Jumonji and restoring the proliferation capacity of T cells. Furthermore, MSC-Luc could selectively migrate to tumor site in a BALB/c nude mouse model with Raji cells. And mice injected with MSC-Tandab in combination with D-1MT significantly inhibited the tumor growth.ConclusionsThese results suggest that UC-MSCs releasing Tandab (CD3/CD19) is an efficient therapeutic tool for the treatment of B cell lymphoma when combined with D-1MT.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-017-0397-z) contains supplementary material, which is available to authorized users.

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Section: Methodsmentioning

confidence: 99%

Mesenchymal stromal cells as vehicles of tetravalent bispecific Tandab (CD3/CD19) for the treatment of B cell lymphoma combined with IDO pathway inhibitor d-1-methyl-tryptophan

et al. 2017

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“…However, this method is not favored clinically due to certain limitations, including the use of radioactive reagents. Over recent years, flow cytometry-based assays have been developed in an attempt to avoid the problems associated with the 51 Cr-assay ( 28 – 29 ). Flow cytometric methods enable rapid analysis to be conducted at the single cell level and may be more convenient for routine application rather than indirectly by the release of a preloaded marker.…”

Section: Discussionmentioning

confidence: 99%

Rapid flow cytometry-based assay for the evaluation of γδ T cell-mediated cytotoxicity

et al. 2017

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The effector function of natural killer, lymphokine-activated killer cells and T lymphocytes is commonly evaluated by radioactive chromium-release cytotoxicity assays. In addition to this indirect method, fluorescence assays have been described for the assessment of in vitro cell-mediated cytotoxicity. In the present study, target cells were stained with 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE), which is a stable integrated fluorescent probe that allows target and effector cells to be distinguished from one another. Staining of target THP-1 cells with 8 µM CFSE revealed high and stable loading of fluorescence and no effect of the viability of cells. After 4 h of in vitro co-culture between γδ T cells and CFSE-labeled infected or uninfected THP-1 cells, staining with propidium iodide (PI) was performed to distinguish between vital and dead cells. During sample acquisition, target cells were gated on the CFSE positivity and examined for cell death based on the uptake of PI. CFSE and PI double positive cells were recognized as the dead target cells. The percentage of cytotoxicity in the CFSE-gated cell population was calculated by subtracting the value obtained for non-specific PI-positive target cells, which was measured in a control group that did not contain effector cells. The present study describes a simple and convenient assay that is based on the direct quantitative and qualitative analysis of cell damage at a single cell level utilizing a two-color flow cytometric assay. In conclusion, the flow cytometric-based assay described in the current study is a simple, sensitive and reliable tool to determine the cytolytic activity of γδ T lymphocytes against mycobacteria. Therefore, the present study may provide valuable information concerning the methods employed to investigate the function of γδ T cells and potentially other lymphocyte subsets.

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“…The monoclonal antibodies used to mark the target cells are anti-CD19, anti-CD33, and anti-CD58. AnnexinV (AnnV) is used to measure apoptosis in the target cell; and 7-AAD or PI for necrosis (28)(29)(30).…”

Section: Monoclonal Antibody Marking Of Target and Effector Cell In Fmentioning

confidence: 99%

Cell-Mediated Cytotoxicity Assays

Özdemir¹

2019

Asthma Allergy Immunol

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Cell-mediated cytotoxicity measurements can be divided in two methods, depending on whether radioactive material is used or not. Natural Killer cell-mediated cytotoxicity is routinely measured with a short-term assay by labeling the target cells with radioactive chromium (51Cr). The advantages of this method are (1) highly sensitivity, (2) easy execution, (3) low spontaneous release, and (4) nontoxic markers. The disadvantages include short half-life of the label and handling and disposal of radioactive supplies. Many attempts have been made to adapt this cytotoxicity assay to abolish radioactivity while maintaining its high sensitivity. Consequently, various nonradioactive methods have been developed. One of these methods utilizing the release of enzymes (LDH) as a result of cytolysis or membrane dyes (PKH-26) is usually less sensitive than radioactive assays. MTT-based colorimetric/enzymatic assays are highly sensitive and easy to use but unfortunately works only with adherent tumor cell targets. Fluorescent dyes such as carboxy-fluorescein diacetate can easily accumulate in the cytoplasm of effector or target cells. After cytotoxicity, the release of the dyes into the supernatant or their retention in target cells is calculated. However, spontaneous release of these dyes can be quite high, causing false positive results leading to a decreased sensitivity and restricting their use in short-term assays. More recently, flow cytometric methods using fluorescent monoclonal antibodies such as anti-CD56 for effector and anti-CD33 for target cells have been defined.

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Single-colour flow cytometric assay to determine NK cell-mediated cytotoxicity and viability against non-adherent human tumor cells

Cited by 6 publications

References 15 publications

Mesenchymal stromal cells as vehicles of tetravalent bispecific Tandab (CD3/CD19) for the treatment of B cell lymphoma combined with IDO pathway inhibitor d-1-methyl-tryptophan

Mesenchymal stromal cells as vehicles of tetravalent bispecific Tandab (CD3/CD19) for the treatment of B cell lymphoma combined with IDO pathway inhibitor d-1-methyl-tryptophan

Rapid flow cytometry-based assay for the evaluation of γδ T cell-mediated cytotoxicity

Cell-Mediated Cytotoxicity Assays

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