Key Points Donor-derived anti-CD19-CAR T cells cause regressions of refractory malignancies after allogeneic transplantation.

A cell undergoing apoptosis demonstrates multitude of characteristic morphological and biochemical features, which vary depending on the inducer of apoptosis, cell type and the “time window” at which the process of apoptosis is observed. Because the gross majority of apoptotic hallmarks can be revealed by flow and image cytometry, the cytometric methods become a technology of choice in diverse studies of cellular demise. Variety of cytometric methods designed to identify apoptotic cells, detect particular events of apoptosis and probe mechanisms associated with this mode of cell death have been developed during the past two decades. In the present review, we outline commonly used methods that are based on the assessment of mitochondrial transmembrane potential, activation of caspases, DNA fragmentation, and plasma membrane alterations. We also present novel developments in the field such as the use of cyanine SYTO and TO-PRO family of probes. Strategies of selecting the optimal multiparameter approaches, as well as potential difficulties in the experimental procedures, are thoroughly summarized.

Amine-reactive N-hydroxysuccinimidyl esters of Alexa Fluor fluorescent dyes with principal absorption maxima at about 555 nm, 633 nm, 647 nm, 660 nm, 680 nm, 700 nm, and 750 nm were conjugated to antibodies and other selected proteins. These conjugates were compared with spectrally similar protein conjugates of the Cy3, Cy5, Cy5.5, Cy7, DY-630, DY-635, DY-680, and Atto 565 dyes. As N-hydroxysuccinimidyl ester dyes, the Alexa Fluor 555 dye was similar to the Cy3 dye, and the Alexa Fluor 647 dye was similar to the Cy5 dye with respect to absorption maxima, emission maxima, Stokes shifts, and extinction coefficients. However, both Alexa Fluor dyes were significantly more resistant to photobleaching than were their Cy dye counterparts. Absorption spectra of protein conjugates prepared from these dyes showed prominent blue-shifted shoulder peaks for conjugates of the Cy dyes but only minor shoulder peaks for conjugates of the Alexa Fluor dyes. The anomalous peaks, previously observed for protein conjugates of the Cy5 dye, are presumably due to the formation of dye aggregates. Absorption of light by the dye aggregates does not result in fluorescence, thereby diminishing the fluorescence of the conjugates. The Alexa Fluor 555 and the Alexa Fluor 647 dyes in protein conjugates exhibited significantly less of this self-quenching, and therefore the protein conjugates of Alexa Fluor dyes were significantly more fluorescent than those of the Cy dyes, especially at high degrees of labeling. The results from our flow cytometry, immunocytochemistry, and immunohistochemistry experiments demonstrate that protein-conjugated, long-wavelength Alexa Fluor dyes have advantages compared to the Cy dyes and other long-wavelength dyes in typical fluorescence-based cell labeling applications.

Mouse thymocytes readily undergo apoptosis-associated DNA degradation upon exposure to glucocorticoids or ionizing radiation. It has been previously shown that flow cytometric cell cycle analysis of propidium iodide-stained apoptotic thymocytes results in the appearance of a distinct cell cycle region (the A, region) below the GdGl region. Cells in this region were shown to be undergoing apoptosis, and determination of apoptosis by flow cytometric analysis was proposed as a superior method for evaluating thymocyte apoptosis. In this study, a variety of DNA binding dyes with diverse primary binding mechanisms were evaluated for their ability to detect glucocorticoid and ionizing radiation-induced apoptosis in mouse thymocytes. Apoptotic thymocytes stained with DNA binding dyes from the phenanthridinium, acridine, actinomycin, chromomycinone, anthracycline, and bisbenzimidazole groups all demonstrated clearly defined A, , regions with percentages comparable to those obtained for propidium iodide. These results indicate that the appearance of the A, region is not dependent on a particular dye binding characteristic and may be the consequence of extensive changes in chromatin structure resulting in a significant degree of dye exclusion.Key terms: Programmed cell death, phenanthridinium, acridine, chromomycinone, anthracycline, bisbenzimidazole Apoptosis, or programmed cell death, is a well-documented phenomenon in many cellular systems (31). Apoptosis is characterized by a number of structural changes in the cell, including the degradation of nuclear chromatin into internucleosomal fragments, presumably by the activation of an endogenous endonuclease (32). Glucocorticoid-(5,32) and ionizing radiation-(28) induced apoptosis in mouse thymocytes has been particularly well characterized in this regard. Internucleosomal DNA fragmentation has formed the basis for the majority of assay systems used to detect apoptosis, including electrophoresis of internucleosoma1 DNA fragments (32) and the separation of small internucleosomal DNA fragments by high-speed centrifugation (33). As previously described, assay systems of this type are by their very nature inadequate since they fail to evaluate apoptosis on a cell by cell basis (27).In a previous paper we described a method by which glucocorticoid-induced apoptosis could be detected in individual, intact mouse thymocytes by reduced fluorescence of the DNA binding dye propidium iodide in the apoptotic subpopulation (27). The apoptotic subpopulation manifested itself as a discrete peak displaying reduced fluorescence with respect to the G$G, cell cycle region. Reduced DNA binding dye fluorescence in apoptotic cells has also been observed using acridine orange (6,12,20), mithramycidethidium bromide (11, and the Hoechst dyes (13,171 in several systems. The apparent ability of DNA binding dyes to distinguish apoptotic cells based on reduced fluorescence by an as yet unclear mechanism has the potential to be an important means of studying programmed cell death,

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The ability of glucocorticoids to induce apoptosis or programmed cell death in mouse thymocytes is well-established. Measurement of apoptosis-associated internucleosomal DNA fragmentation through determination of the percentage of fragmented DNA by electrophoresis or centrifugation of whole cell lysates is by far the most common means of quantifying apoptosis. Since these methods measure DNA fragmentation in whole cell lysates rather than intact cells, they have severe limitations, particularly with heterogeneous cell populations. When mouse thymocytes were incubated with glucocorticoids, fixed, stained with propidium iodide and analysed flow cytometrically for cell cycle distribution, a distinct subpopulation of cells was observed to form below the G0/G1 region, denoted as the A0 region. The presence of cells in this region was consistent with the presence of internucleosomal DNA fragments as determined by gel electrophoresis. Inhibitors of transcription, translation and endonuclease activity, and a glucocorticoid receptor antagonist prevented accumulation of cells in this region. Irradiation of mouse thymocytes also produced a population in the A0 region. Cells in this region are believed to have undergone glucocorticoid-induced DNA fragmentation. This method represents a useful alternative to whole cell lysate assays, since apoptosis can be evaluated on an individual cell basis.

To facilitate the study of signaling pathways involved in myeloid dendritic cell (DC) differentiation, we have developed a serum-free culture system in which human CD14+ peripheral blood monocytes differentiate rapidly in response to bacterial LPS, TNF-α, or calcium ionophore (CI). Within 48–96 h, depending on the inducing agent, the cells acquire many immunophenotypical, morphological, functional, and molecular properties of DC. However, there are significant differences in the signaling pathways used by these agents, because 1) LPS-induced, but not CI-induced, DC differentiation required TNF-α production; and 2) cyclosporin A inhibited differentiation induced by CI, but not that induced by LPS. Nevertheless, all three inducing agents activated members of the NF-κB family of transcription factors, including RelB, suggesting that despite differences in upstream elements, the signaling pathways all involve NF-κB. In this report we also demonstrate and offer an explanation for two observed forms of the RelB protein and show that RelB can be induced in myeloid cells, either directly or indirectly, through a calcium-dependent and cyclosporin A-sensitive pathway.