A real‐time digital bio‐imaging system to quantify cellular cytotoxicity as an alternative to the standard chromium‐51 release assay

Abstract

Reliable measurement of cellular cytotoxicity is essential for the characterization of immune responses and for the monitoring of antibody treatment efficacy. Until now, the standard Cr-release assay has remained the sole sensitive assay that measures cellular cytotoxicity. Alternative non-radioactive assays have been developed but they do not provide accurate measurement of target cell cytotoxicity. The cost and hazard of handling radioactivity are strong incentives to find alternative solutions to Cr. We took advantage of the recent development of cell-imaging multimode readers to develop a novel non-radioactive and real-time cytotoxic assay that demonstrates good reproducibility and sensitivity. The extent of target-cell cytotoxicity is monitored over time by imaging and quantifying live fluorescent target cells in 96-well plates. We have developed classical natural killer cell assays in the presence or absence of blocking antibodies and antibody-dependent cell-mediated cytotoxicity. We show that in these assays, cell killing occurs within the first 2 hr with half maximum killing reached after 30 min. This technology has numerous applications such as natural killer and T-cell cytotoxicity assays and can be extended to cell survival and apoptosis measurement assays.

A ganglioside-based senescence-associated immune checkpoint

“…A real-time cytotoxic assay was performed as previously described 22,23 . Briefly, target cells were labelled with 0.5µM Calcein-AM (Molecular Probes) for 15 min at room temperature.…”

New Plant Growth-Promoting, Chromium-Detoxifying Microbacterium Species Isolated From a Tannery Wastewater: Performance and Genomic Insights

“…A positive control was used by inoculating bacterial suspension on agar plates without metal. The half maximal inhibitory concentration (IC 50 ) was determined using experiments conducted in tryptic soy broth (TSB) medium at 30°C with shaking at 120 rpm (Fassy et al, 2017 ). Bacterial growth was measured after 24 h on a UV–visible (UV-Vis) spectrophotometer at 600 nm.…”

Rationale for combination of therapeutic antibodies targeting tumor cells and immune checkpoint receptors: Harnessing innate and adaptive immunity through IgG1 isotype immune effector stimulation

“…Experimentally, ex vivo and in vitro assays with patients’ purified lymphocyte populations [ 3 , 19 , 25 , 26 , 41 ] from the tumor microenvironment and the peripheral blood are used to directly observe NK cell activation and lytic activity [ 31 , 42 , 43 ]. Indirect measurements are performed using markers on circulating and tumor-infiltrating T cells, NK cells, and DCs, as well as cytokine levels in the plasma [ 1 , 44 ], including expression of activating receptors such as CD16, CD107a, CD137, NK group 2 member D (NKG2D), and NK cell p46–related protein (NKp46) receptors [ 26 , 42 , 45 ].…”