Flow cytometric screening of cell-penetrating peptides for their uptake into embryonic and adult stem cells

Manceur, Aziza; Wu, A. M.; Audet, Julie

doi:10.1016/j.ab.2007.02.015

Cited by 35 publications

(24 citation statements)

References 38 publications

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“…To remove the peptides associated with the outer plasma membrane, we used trypan blue, rather than the moretypical trypsin, 16 to wash all of the cells prior to analysis; trypsin is not an e®ective reagent to digest outer-membrane-bound oligoarginines containing D-arginine residues, whereas trypan blue (500 g/ mL) can quench more than 80% of the cell-surface FITC emission and has been used widely in previous studies. [30][31][32] Although R 8 exhibited the most di®use pattern with a small amount of vesicle staining at a high concentration (10 M), most of the FITC-R 8 molecules were trapped in punctate structures, with no signi¯cant cytosolic di®usion or nuclear accumulation at concentrations ranging from 2.5 to 5 M (see Fig. 1), in good agreement with the distributions described by others.…”

Section: The Intracellular Distribution Of Octa-d-arginine (R 8 )supporting

confidence: 78%

The backbone stereochemistry influences the intracellular distribution and uptake mechanism of oligoarginines

Chen

et al. 2014

J. Innov. Opt. Health Sci.

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D-arginine oligomers have been widely used as intracellular delivery vectors both in in vitro and in vivo application. Nevertheless, their internalization pathway is obscure and con°icting results have been obtained concerning their intracellular distribution. In this study, we demonstrate that octa-D-arginine (r 8 ) undergoes di®use localization throughout the cytoplasm and nucleus even at low concentrations and that r 8 (r: D-arginine) enters the cells via direct membrane translocation, unlike R 8 (R: L-arginine), of which endocytosis is the major internalization pathway. The observation that R 8 and r 8 enter the cells through two clearly distinct internalization pathways suggests that the backbone stereochemistry a®ects the uptake mechanism of oligoarginines.

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Section: The Intracellular Distribution Of Octa-d-arginine (R 8 )supporting

confidence: 78%

The backbone stereochemistry influences the intracellular distribution and uptake mechanism of oligoarginines

Chen

et al. 2014

J. Innov. Opt. Health Sci.

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“…The cells were washed with PBS, and then the peptide/Hoechst 33342 mixtures (300 μL) were gently added to the cells. After incubation for 5 or 30 min at 37°C, the supernatant was discarded, and a solution of TB (200 μL, 500 μg/mL) in PBS was added to quench the extracellular fluorescence from the outer membrane-bound peptides [31,43,44]. After 1 min, the cells were washed with PBS containing propidium iodide (PI, 2.5 μg/mL) and analyzed live in serum-free DMEM (250 μL).…”

Section: Confocal Laser Scanning Microscopy (Clsm)mentioning

confidence: 99%

Direct cytosolic delivery of cargoes in vivo by a chimera consisting of D- and L-arginine residues

Chen

et al. 2012

Journal of Controlled Release

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“…Before analysis, the samples are transferred to FACS tubes containing trypan blue (TB) for analyzing intracellular accumulation, and/or propidium iodide (PI) for analysis of membrane integrity (optionally). TB is a quencher that is confined to the outside of living cells and effectively eliminates FAM-fluorescence by concentration quenching (17,18). The remaining fluorescence arises from the intracellular-located FAM-CPP that is inaccessible to TB.…”

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confidence: 99%

Uptake Kinetics of Cell-Penetrating Peptides

Florén

Mäger

Langel

2010

Methods in Molecular Biology

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As our knowledge increases about the diversity in uptake mechanisms displayed by cell-penetrating peptides (CPP), the concept of CPP uptake kinetics becomes increasingly complex. Here, we present three different assays that can be used for studying different kinetic aspects of CPP-mediated delivery: intracellular accumulation and membranolytical effects, intracellular CPP-cargo detachment, and finally a functional readout of a biological action from the delivered cargo. Unlike the traditional end-point measurements that give a static postincubation readout, these assays are all dynamic, real-time, in situ measurements obtained during incubation. A combination of some (or all) of these different assays gives us not only interesting kinetic information about the uptake routes but also provides a simple and valuable methodology for the evaluation of potential drug candidates based on the chemical modification of CPPs by cargo attachment.

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Flow cytometric screening of cell-penetrating peptides for their uptake into embryonic and adult stem cells

Cited by 35 publications

References 38 publications

The backbone stereochemistry influences the intracellular distribution and uptake mechanism of oligoarginines

The backbone stereochemistry influences the intracellular distribution and uptake mechanism of oligoarginines

Direct cytosolic delivery of cargoes in vivo by a chimera consisting of D- and L-arginine residues

Uptake Kinetics of Cell-Penetrating Peptides

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