Microwell-Based Live Cell Imaging of NK Cell Dynamics to Assess Heterogeneity in Motility and Cytotoxic Response

Vanherberghen, Bruno; Frisk, Thomas; Forslund, Elin; Olofsson, Per; Guldevall, Karolin; Önfelt, Björn

doi:10.1007/978-1-4939-3684-7_8

Cited by 4 publications

(4 citation statements)

References 10 publications

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“…Calcein labels the cytosol of viable cells making them clearly detectable by fluorescence microscopy. Upon cell death Calcein green leaks out making it possible to monitor NK cell induced target cell death through loss of fluorescence 34 .…”

Section: Methodsmentioning

confidence: 99%

A collagen-based microwell migration assay to study NK-target cell interactions

Olofsson

Brandt

Magnusson

et al. 2019

Sci Rep

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Natural killer (NK) cell cytotoxicity in tissue is dependent on the ability of NK cells to migrate through the extracellular matrix (ECM) microenvironment. Traditional imaging studies of NK cell migration and cytotoxicity have utilized 2D surfaces, which do not properly reproduce the structural and mechanical cues that shape the migratory response of NK cells in vivo . Here, we have combined a microwell assay that allows long-term imaging and tracking of small, well-defined populations of NK cells with an interstitial ECM-like matrix. The assay allows for long-term imaging of NK–target cell interactions within a confined 3D volume. We found marked differences in motility between individual cells with a small fraction of the cells moving slowly and being confined to a small volume within the matrix, while other cells moved more freely. A majority of NK cells also exhibited transient variation in their motility, alternating between periods of migration arrest and movement. The assay could be used as a complement to in vivo imaging to study human NK cell heterogeneity in migration and cytotoxicity.

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Section: Methodsmentioning

confidence: 99%

A collagen-based microwell migration assay to study NK-target cell interactions

Olofsson

Brandt

Magnusson

et al. 2019

Sci Rep

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“…Additionally, very fast migratory events, such as Natural Killer (NK) cell 45 and Dendritic Cell (DC) 46 movement, may be missed by imaging every 15 min. However, imaging intervals can be optimized to study the specific cell-cell interactions of interest, and the ability to image in 3 dimensions through z-stacks allows the assessment of 3D cell movement.…”

Section: Discussionmentioning

confidence: 99%

Interrogating Cell-Cell Interactions in the Salivary Gland <em>via Ex Vivo</em> Live Cell Imaging

Elder,

Cholewa-Waclaw,

Emmerson

2023

JoVE

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Salivary gland regeneration is a complex process involving intricate interactions among various cell types. Recent studies have shed light on the pivotal role played by macrophages in driving the regenerative response. However, our understanding of this critical role has primarily relied on static views obtained from fixed tissue biopsies.To overcome this limitation and gain insights into these interactions in real time, this study outlines a comprehensive protocol for culturing salivary gland tissue ex vivo and capturing live images of cell migration.The protocol involves several key steps: First, mouse submandibular salivary gland tissue is carefully sliced using a vibratome and then cultured at an air-liquid interface.These slices can be intentionally injured, for instance, through exposure to radiation, to induce cellular damage and trigger the regenerative response. To track specific cells of interest, they can be endogenously labeled, such as by utilizing salivary gland tissue collected from genetically modified mice where a particular protein is marked with green fluorescent protein (GFP). Alternatively, fluorescently-conjugated antibodies can be employed to stain cells expressing specific cell surface markers of interest. Once prepared, the salivary gland slices are subjected to live imaging using a high-content confocal imaging system over a duration of 12 h, with images captured at 15 min intervals. The resulting images are then compiled to create a movie, which can subsequently be analyzed to extract valuable cell behavior parameters.This innovative method provides researchers with a powerful tool to investigate and better understand macrophage interactions within the salivary gland following injury, thereby advancing our knowledge of the regenerative processes at play in this dynamic biological context.

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“…Time lapse cinematography indicated that, in NK modes without antibody, there is heterogeneity in serial killing. Some cells within the effector cell populations bound to targets without killing them while the cytotoxic cells killed 1, 2, 3, or even up to 5-7 times [16,21]. Microchip experiments also indicated cellular differences in killing when the NK cells are in ADCC mode.…”

Section: Introductionmentioning

confidence: 98%

Natural Killer (NK) Cell Expression of CD2 as a Predictor of Serial Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)

et al. 2020

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NK cell ADCC supports monoclonal antibody anti-tumor therapies. We investigated serial ADCC and whether it could be predicted by NK phenotypes, including expression of CD16A, CD2 and perforin. CD16A, the NK receptor for antibodies, has AA158 valine or phenylalanine variants with different affinities for IgG. CD2, a costimulatory protein, associates with CD16A and can augment CD16A-signaling. Pore-forming perforin is essential for rapid NK-mediated killing. NK cells were monitored for their ADCC serial killing frequency (KF). KF is the average number of target cells killed per cell by a cytotoxic cell population. KF comparisons were made at 1:4 CD16pos NK effector:target ratios. ADCC was toward Daudi cells labeled with 51Cr and obinutuzumab anti-CD20 antibody. CD16A genotypes were determined by DNA sequencing. CD2, CD16A, and perforin expression was monitored by flow cytometry. Serial killing KFs varied two-fold among 24 donors and were independent of CD16A genotypes and perforin levels. However, high percentages of CD2pos of the CD16Apos NK cells and high levels of CD16A were associated with high KFs. ROC analysis indicated that the %CD2pos of CD16Apos NK cells can predict KFs. In conclusion, the extent of serial ADCC varies significantly among donors and appears predictable by the CD2posCD16Apos NK phenotype.

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Microwell-Based Live Cell Imaging of NK Cell Dynamics to Assess Heterogeneity in Motility and Cytotoxic Response

Cited by 4 publications

References 10 publications

A collagen-based microwell migration assay to study NK-target cell interactions

A collagen-based microwell migration assay to study NK-target cell interactions

Interrogating Cell-Cell Interactions in the Salivary Gland <em>via Ex Vivo</em> Live Cell Imaging

Natural Killer (NK) Cell Expression of CD2 as a Predictor of Serial Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)

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