Computational protocol: Melanoma cell lysosome secretory burst neutralizes the CTL-mediated cytotoxicity at the lytic synapse

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Protocol publication

[…] Images were scored by evaluating, for each experimental condition, at least 45 CTL/target cell conjugates in randomly selected fields from at least three independent experiments. GrzB staining was evaluated by scoring the percentage of positive target cells. To quantify the polarization of CD63+ vesicles towards the immunological synapse or synaptic enrichment of Av-SRho staining, unprocessed images were analysed by dividing each target cell into three equal regions. Fluorescence was measured using the ROI statistics tool of the ImageJ software on image projections in two equally defined volumes (one at the synaptic area and the other at the distal area). To exclude CD63 or Av-SRho staining of CTL, a region was carefully drawn on the melanoma cell side of the lytic synapse. Results were reported as the fold increase of the corresponding staining integrated FI at the synaptic region divided by the same FI measured at the distal region (see schemes drawn in ). For quantification of perforin on melanoma cells, the lytic synapse was divided into two equal volumes, one on the CTL and one on the melanoma cell side. Total perforin FI at the lytic synapse of both volumes was acquired on image projections of CTL/melanoma conjugates. Perforin on melanoma cells was quantified as the percentage of perforin FI at the defined melanoma cell volume (red box) divided by the totality of perforin FI (black box) at the lytic synapse (% of perforin FI=(perforin FI on melanoma cell/total perforin FI) × 100; see schemes drawn in ) using the ImageJ software. The above-described measurements were confirmed by analysing FI in melanoma cells z-stack projections using the Region Measurements of the Metamorph software (Universal Imaging). In further experiments z-stack images of CTL interacting with melanoma cells were reconstructed and analysed using the Surface tool of the Imaris software. […]

Pipeline specifications

Software tools ImageJ, MetaMorph, Imaris
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Homo sapiens
Diseases Melanoma, Neoplasms, Drug-Related Side Effects and Adverse Reactions