A simple and sensitive flow cytometric assay for the determination of the cytotoxic activity of human natural killer cells

Radošević, Katarina; Garritsen, Henk; Graft, M. van; Grooth, B.G. de; Greve, Jan

doi:10.1016/0022-1759(90)90259-x

Cited by 59 publications

(21 citation statements)

References 8 publications

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“…Here, Tat might exert an antimicrobial activity before being degraded by cell-released proteases. Previous reports have identified Tat (47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58) as an antimicrobial peptide with broadspectrum antibacterial activity against many Gram-positive and Gram-negative bacteria, including S. aureus, E. coli, and S. Typhimurium (65,82). We also observed a marked bactericidal effect of Tat against E. coli K1 RS218 and partly against Shigella but not against Salmonella in functionality assays (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…During the ongoing incubation, samples were taken at each time point, 0.2% trypan blue was added, and samples were directly analyzed by flow cytometry using a BD FACSCalibur flow cytometer (BD Biosciences, Heidelberg, Germany). To monitor potential deleterious effects of CPP-FITC on membrane integrity, 1 g/ml propidium iodide (PI) was added during coincubation with CPP-FITC, and the fluorescence of intracellular PI was recorded by flow cytometry as a measure of membranolysis (47). As a positive control for disruption of membrane integrity, 0.2% Triton X-100 (AppliChem, Darmstadt, Germany) was added before the measurements.…”

Section: Methodsmentioning

confidence: 99%

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Bacterium-Derived Cell-Penetrating Peptides Deliver Gentamicin To Kill Intracellular Pathogens

Gomarasca

Martins

Greune

et al. 2017

Antimicrob Agents Chemother

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Commonly used antimicrobials show poor cellular uptake and often have limited access to intracellular targets, resulting in low antimicrobial activity against intracellular pathogens. An efficient delivery system to transport these drugs to the intracellular site of action is needed. Cell-penetrating peptides (CPPs) mediate the internalization of biologically active molecules into the cytoplasm. Here, we characterized two CPPs, ␣1H and ␣2H, derived from the Yersinia enterocolitica YopM effector protein. These CPPs, as well as Tat (trans-activator of transcription) from HIV-1, were used to deliver the antibiotic gentamicin to target intracellular bacteria. The YopM-derived CPPs penetrated different endothelial and epithelial cells to the same extent as Tat. CPPs were covalently conjugated to gentamicin, and CPP-gentamicin conjugates were used to target infected cells to kill multiple intracellular Gram-negative pathogenic bacteria, including Escherichia coli K1, Salmonella enterica serovar Typhimurium, and Shigella flexneri. Taken together, CPPs show great potential as delivery vehicles for antimicrobial agents and may contribute to the generation of new therapeutic tools to treat infectious diseases caused by intracellular pathogens.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Bacterium-Derived Cell-Penetrating Peptides Deliver Gentamicin To Kill Intracellular Pathogens

Gomarasca

Martins

Greune

et al. 2017

Antimicrob Agents Chemother

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“…K562 cells, for use as targets, were grown as previously described (Rado~evi6 et al, 1990). The NK cell-clone NK76 was generously provided by Dr. R.L.H.…”

Section: Cellsmentioning

confidence: 99%

A flow cytometric study of the membrane potential of natural killer and K562 cells during the cytotoxic process

Radošević

Schut

Graft

et al. 1993

Journal of Immunological Methods

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This study demonstrates that it is possible to investigate the membrane potential of interacting cells during the cytotoxic process using flow cytometry. Changes in the membrane potential of NK and K562 cells, involved in a cell-mediated cytotoxic process, were studied by standard and slit-scan flow cytometry, using the membrane potential sensitive fluorescent probe DiBAC4(3). The NK cells were labeled with a membrane marker (TR-18 or DiI) prior to incubation with K562 cells and the conjugates that were formed could be identified on the basis of the membrane marker fluorescence and light scattering signals. With a slit-scan technique we measured the membrane potential of each cell in a conjugate separately. The results show that depolarization of the K562 cell occurs as a consequence of the cytotoxic activity of the NK cell. This depolarization appears to be an early sign of cell damage because the cell membrane still remains impermeable to propidium iodide. Our data also indicate that depolarization of the NK cell occurs as a result of its cytotoxic activity.

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“…To study these possibilities immobile NK cells loaded with F-18 were used. From previous studies (Rado~evic et al, 1990) we know that leakage of this dye could be neglected. A typical result is shown in Fig.…”

Section: Time-dependent Changes Of Fluorochrome Signals In Unstimulatmentioning

confidence: 99%

A simple optical fiber device for quantitative fluorescence microscopy of single living cells

Graft

Oosterhuis

Werf

et al. 1993

Journal of Immunological Methods

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A simple and relatively inexpensive system is described for obtaining quantitative fluorescence measurements on single living cells loaded with a fluorescent probe to study cell physiological processes. The light emitted from the fluorescent ceils is captured by and transported through an optical fiber. After passage through appropriate filters the light is measured using a photomultiplier tube. The optical fiber is mounted in one of the microscope outlets. Signals derived from the photomultiplier are converted to voltage, amplified, and displayed on a recorder. In the excitation pathway a shutter control unit is mounted. With this control unit the period that the excitation pathway is 'opened' and 'closed' can be adjusted, to reduce cell damage and/or bleaching of the probe. This option allows time-lapse recording of experiments up to 1 h. We have used this set-up with a single and dual emission fluorescent probe to determine intracellular calcium concentrations and pH, respectively. In Fluo-3-1oaded K562 target ceils bound to natural killer cells, a temporary rise in [Ca2+]i was accompanied by bleb formation. The simple construction of this set-up is interchangeable between different types of fluorescence microscopes and can easily be combined with other microscopy techniques, e.g., patch clamp.

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A simple and sensitive flow cytometric assay for the determination of the cytotoxic activity of human natural killer cells

Cited by 59 publications

References 8 publications

Bacterium-Derived Cell-Penetrating Peptides Deliver Gentamicin To Kill Intracellular Pathogens

Bacterium-Derived Cell-Penetrating Peptides Deliver Gentamicin To Kill Intracellular Pathogens

A flow cytometric study of the membrane potential of natural killer and K562 cells during the cytotoxic process

A simple optical fiber device for quantitative fluorescence microscopy of single living cells

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