NK Cell-Mediated Cytotoxicity Assay

After stimulation, NK cells were co-cultured with YAC-1 target cells (mouse lymphoma lymphoblast fibroblast; ATCC TIB-160; authenticated and mycoplasma tested) at the indicated effector cell:target cell (E:T) ratios for 4 h (37 °C and 5% CO₂). Co-cultures were then stained with Fixable Viability Dye™ (FVD) eFluor™ 506 (eBioscience) and fluorochrome-conjugated antibodies specific for cell surface markers (CD45, CD3 and NK1.1) to differentiate NK and YAC-1 cells and analyzed by flow cytometry. NK cell only and YAC-1 only controls were used to determine spontaneous lysis of target cells and set gating strategy. Percent specific lysis was calculated as the proportion of YAC-1 cells (FSC^int SSC^int-hi CD45⁺ CD3⁺ CD49b^-) positive for FVD, minus spontaneous lysis (Littwitz-Salomon et al, 2018 (link); Valiathan et al, 2012 (link)).

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Mayall J.R., Horvat J.C., Mangan N.E., Chevalier A., McCarthy H., Hampsey D., Donovan C., Brown A.C., Matthews A.Y., de Weerd N.A., de Geus E.D., Starkey M.R., Kim R.Y., Daly K., Goggins B.J., Keely S., Maltby S., Baldwin R., Foster P.S., Boyle M.J., Tanwar P.S., Huntington N.D., Hertzog P.J, & Hansbro P.M. (2024). Interferon-epsilon is a novel regulator of NK cell responses in the uterus. EMBO Molecular Medicine, 16(2), 267-293.

Publication 2024

Fixable Viability Dye™ (FVD) eFluor™ 506

Corresponding Organization :

Other organizations : Hunter Medical Research Institute, Hudson Institute of Medical Research, Monash University, Centenary Institute, University of Technology Sydney, John Hunter Hospital, University of Newcastle Australia, Australian Regenerative Medicine Institute

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Variable analysis

independent variables

Effector cell:target cell (E:T) ratios

dependent variables

Percent specific lysis of YAC-1 cells

control variables

Co-culture temperature (37 °C)
Co-culture atmosphere (5% CO2)
Co-culture duration (4 h)

controls

Positive control: NK cell only control to determine spontaneous lysis of target cells
Negative control: YAC-1 only control to set gating strategy

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