Multiplexed quantification of autophagic flux by imaging flow cytometry.

Imaging flow cytometry allows for the quantitative assessment of fluorescent signals at the subcellular level. Here, we describe the use of a biosensor cell line, namely, U2OS osteosarcoma cells equipped with a fusion protein comprising monomeric red fluorescent protein (mRFP), green fluorescent protein (GFP) and microtubule-associated proteins 1A/1B light chain 3B (best known as LC3), for the assessment of autophagic flux by imaging flow cytometry. We detail all analysis tools required to distinguish autophagosomes (that emit both a red and a green fluorescence) and autolysosomes (that emit a red fluorescence, yet lose the green fluorescent signal) and to quantitate autophagic flux in a convenient fashion.

Quantification of cellular viability by automated microscopy and flow cytometry.

Cellular viability is usually determined by measuring the capacity of cells to exclude vital dyes such as 4',6-diamidino-2-phenylindole (DAPI), or by assessing nuclear morphology with chromatinophilic plasma membrane-permeant dyes, such as Hoechst 33342. However, a fraction of cells that exclude DAPI or exhibit normal nuclear morphology have already lost mitochondrial functions and/or manifest massive activation of apoptotic caspases, and hence are irremediably committed to death. Here, we developed a protocol for the simultaneous detection of plasma membrane integrity (based on DAPI) or nuclear morphology (based on Hoechst 33342), mitochondrial functions (based on the mitochondrial transmembrane potential probe DiOC6(3)) and caspase activation (based on YO-PRO®-3, which can enter cells exclusively upon the caspase-mediated activation of pannexin 1 channels). This method, which allows for the precise quantification of dead, dying and healthy cells, can be implemented on epifluorescence microscopy or flow cytometry platforms and is compatible with a robotized, high-throughput workflow.

Screening of novel immunogenic cell death inducers within the NCI Mechanistic Diversity Set.

Immunogenic cell death (ICD) inducers can be defined as agents that exert cytotoxic effects while stimulating an immune response against dead cell-associated antigens. When initiated by anthracyclines, ICD is accompanied by stereotyped molecular changes, including the pre-apoptotic exposure of calreticulin (CRT) on the cell surface, the lysosomal secretion of ATP during the blebbing phase of apoptosis, and the release of high mobility group box 1 (HMGB1) from dead cells. By means of genetically engineered human osteosarcoma U2OS cells, we screened the 879 anticancer compounds of the National Cancer Institute (NCI) Mechanistic Diversity Set for their ability to promote all these hallmarks of ICD in vitro. In line with previous findings from our group, several cardiac glycosides exhibit a robust propensity to elicit the major manifestations of ICD in cultured neoplastic cells. This screen pointed to septacidin, an antibiotic produced by Streptomyces fibriatus, as a novel putative inducer of ICD. In low-throughput validation experiments, septacidin promoted CRT exposure, ATP secretion and HGMB1 release from both U2OS cells and murine fibrosarcoma MCA205 cells. Moreover, septacidin-killed MCA205 cells protected immunocompetent mice against a re-challenge with living cancer cells of the same type. Finally, the antineoplastic effects of septacidin on established murine tumors were entirely dependent on T lymphocytes. Altogether, these results underscore the suitability of the high-throughput screening system described here for the identification of novel ICD inducers.

Antiapoptotic activity of argon and xenon.

Although chemically non-reactive, inert noble gases may influence multiple physiological and pathological processes via hitherto uncharacterized physical effects. Here we report a cell-based detection system for assessing the effects of pre-defined gas mixtures on the induction of apoptotic cell death. In this setting, the conventional atmosphere for cell culture was substituted with gas combinations, including the same amount of oxygen (20%) and carbon dioxide (5%) but 75% helium, neon, argon, krypton, or xenon instead of nitrogen. The replacement of nitrogen with noble gases per se had no effects on the viability of cultured human osteosarcoma cells in vitro. Conversely, argon and xenon (but not helium, neon, and krypton) significantly limited cell loss induced by the broad-spectrum tyrosine kinase inhibitor staurosporine, the DNA-damaging agent mitoxantrone and several mitochondrial toxins. Such cytoprotective effects were coupled to the maintenance of mitochondrial integrity, as demonstrated by means of a mitochondrial transmembrane potential-sensitive dye and by assessing the release of cytochrome c into the cytosol. In line with this notion, argon and xenon inhibited the apoptotic activation of caspase-3, as determined by immunofluorescence microscopy coupled to automated image analysis. The antiapoptotic activity of argon and xenon may explain their clinically relevant cytoprotective effects.

Recovery from transient global amnesia following restoration of hippocampal and fronto - cingulate perfusion.

A patient who suffered a transient global amnesia (TGA) attack underwent regional cerebral blood flow (rCBF) SPECT imaging and neuropsychological testing in the acute phase, after one month and after one year. Neuropsychological testing in the acute phase showed a pattern of anterograde and retrograde amnesia, whereas memory was within age normal limits at follow up. SPECT data were analysed with a within subject comparison and also compared with those of a group of healthy controls. Within subject comparison between the one month follow up and the acute phase detected increases in rCBF in the hippocampus bilaterally; further rCBF increases in the right hippocampus were detected after one year. Compared to controls, significant hypoperfusion was found in the right precentral, cingulate and medial frontal gyri in the acute phase; after one month significant hypoperfusion was detected in the right precentral and cingulate gyri and the left postcentral gyrus; after one year no significant hypoperfusion appeared. The restoration of memory was paralleled by rCBF increases in the hippocampus and fronto-limbic-parietal cortex; after one year neither significant rCBF differences nor cognitive deficits were detectable. In conclusion, these data indicate that TGA had no long lasting cognitive and neural alterations in this patient.