Excitation Shift Research Articles

Exploring Unusual Confined Chemistry: Excited-State Proton Transfer Reaction in Supported Liquid Membrane with Deep Eutectic Solvents.

Supported liquid membrane (SLM) incorporating ionic liquids (ILs) or deep eutectic solvents (DESs) offers a promising method for ion and (bio)chemical separations and CO2 capture. However, a molecular understanding of whether chemical reactions occur in these confined media is crucial. We report excited-state proton transfer (ESPT) reaction of a photoacid, HPTS, in various DES-based SLMs (pore size ∼280 nm) using steady-state and time-resolved fluorescence spectroscopy. Our findings reveal that, while the ESPT is unfavorable in bulk DESs, it occurs substantially in SLM-containing DESs. Time-resolved area normalized emission spectra (TRANES) show that ESPT time ranges from 2.6 to 7.5 ns and is greatly affected by changes in DES constituents. The results suggest that HPTS interacts with ordered DES structures formed by long-range interfacial effects in membrane pores, making it suitable for ESPT. Furthermore, it is found that the dynamics of solvent relaxation in confined DESs are significantly slower than in bulk liquids. This observation, together with a large red-edge excitation shift, supports the impact of long-range interfacial effects on the DES structure inside membrane pores. Given the task-specific properties of DESs, the incorporation of these solvents into SLM pores can be a useful strategy for investigating new chemical processes.

Reversible pH-switchable NIR-II nano-photosensitizer for precise imaging and photodynamic therapy of tumors

Photodynamic therapy (PDT) has attracted widespread attention from researchers as an emerging cancer treatment method. There have been many reports on various types of NIR-II photosensitizers for imaging and treatment of tumor sites. However, there are few reports on the development of NIR-II organic small molecule photosensitizers that have intelligent response to the tumor microenvironment, precise imaging, real-time treatment, and high biocompatibility. In this work, we developed a series of NIR-II photosensitizers (RBTs) with near-infrared excitation, good photostability, and large Stokes shift. Among them, RBT-Br exhibited higher reactive oxygen species (ROS) generation efficiency due to the introduction of halogen heavy atoms to enhance intersystem crossing (ISC). It is noteworthy that RBT-Br can generate singlet oxygen (1O2) and superoxide anion radicals (•O2−) simultaneously under 730 nm laser. Subsequently, we used molecular engineering technology to construct three pH-responsive NIR-II photosensitizers (RBT-pHs) by utilizing the closure of the lactam ring, among which RBT-pH-1 (pKa = 6.78) is able to be directionally activated under the stimulation of tumor micro-acid environment, with its fluorescence emission window reaching 933 nm. Subsequently, RBT-pH-1 NPs encapsulated in DSPE-mPEG5k were applied for PDT treatment of mouse tumors. The results showed that RBT-pH-1 NPs were activated by the acidic tumor microenvironment and generated ROS under laser excitation, exhibiting precise tumor imaging and significant tumor growth inhibition. We look forward to these multifunctional NIR-II organic small molecule photosensitizers providing a more efficient approach for clinical treatment of tumors. Statement of significanceA reversible pH-switchable NIR-II nano-photosensitizer RBT-pH-1 NPs (pKa = 6.76) is developed for precise imaging and PDT therapy of mouse tumors, which can be effectively used for targeted enrichment and activation of tumor micro-acid environments. The results show that this NIR-II photosensitizer generates ROS through tumor micro-acid environment stimulation and laser triggering, showing precise tumor imaging guidance and significant tumor growth inhibition.

Excitation signatures of isochorically heated electrons in solids at finite wave number explored from first principles

Ultrafast heating of solids with modern x-ray free electron lasers (XFELs) leads to a unique set of conditions characterized by the simultaneous presence of heated electrons in a cold ionic lattice. In this work, we analyze the effect of electronic heating on the dynamic structure factor (DSF) in bulk aluminum (Al) with a face-centered cubic lattice and in silicon (Si) with a crystal diamond structure using first-principles linear-response time-dependent density functional theory simulations. We find a thermally induced red shift of the collective plasmon excitation in both materials. In addition, we show that the heating of the electrons in Al can lead to the formation of a double-plasmon peak due to the extension of the Landau damping region to smaller wave numbers. Finally, we demonstrate that thermal effects generate a measurable and distinct signature (peak-valley structure) in the DSF of Si at small frequencies. Our simulations indicate a variety of new features in the spectrum of x-ray-driven solids, specifically at finite momentum transfer, which can be probed in upcoming x-ray Thomson scattering experiments at various XFEL facilities. Published by the American Physical Society 2024

The effect of chrysin binding on the conformational dynamics and unfolding pathway of human serum albumin

Studies on the interactions between ligands and proteins provide insights into how a possible medication alters the structures and activities of the target or carrier proteins. The natural flavonoid aglycone Chrysin (CHR) has demonstrated anti-inflammatory, antioxidant, antiapoptotic, neuroprotective, and antineoplastic effects, both in vitro and in vivo. In this work, we investigated the impact of CHR binding on the as-yet-unexplored conformation, dynamics, and unfolding mechanism of human serum albumin (HSA). We determined CHR binding to HSA domain-II with the association constant (Ka) of 2.70 ± 0.21 × 105 M−1. The urea-induced sequential unfolding mechanism of HSA was used to elucidate the debatable binding location of CHR. CHR binding induced both secondary and tertiary structural alterations in the protein as studied by far-UV circular dichroism and intrinsic fluorescence spectroscopy. Red edge excitation shift (REES) indicated a decrease in conformational dynamics of the protein on the complex formation. This suggested an ordered compact and spatial arrangement of the CHR-boundmolecule. The binding of CHR was found to significantly modulate the urea-induced unfolding pathway of HSA. Urea-induced unfolding pathway of HSA became a two-state process (N-U) from a three-state process (N-I-U). The interaction of CHR is found to increase the thermal stability of the protein by ∼4 °C. This study focuses on the fundamental sciences and demonstrates how prospective medication compounds can alter the dynamics and stability of protein structure.

Dopamine D2 Receptor Modulates Exercise Related Effect on Cortical Excitation/Inhibition and Motor Skill Acquisition.

Exercise is known to benefit motor skill learning in health and neurological disease. Evidence from brain stimulation, genotyping, and Parkinson's disease studies converge to suggest that the dopamine D2 receptor, and shifts in the cortical excitation and inhibition (E:I) balance, are prime candidates for the drivers of exercise-enhanced motor learning. However, causal evidence using experimental pharmacological challenge is lacking. We hypothesized that the modulatory effect of the dopamine D2 receptor on exercise-induced changes in the E:I balance would determine the magnitude of motor skill acquisition. To test this, we measured exercise-induced changes in excitation and inhibition using paired-pulse transcranial magnetic stimulation (TMS) in 22 healthy female and male humans, and then had participants learn a novel motor skill-the sequential visual isometric pinch task (SVIPT). We examined the effect of D2 receptor blockade (800 mg sulpiride) on these measures within a randomized, double-blind, placebo-controlled design. Our key result was that motor skill acquisition was driven by an interaction between the D2 receptor and E:I balance. Specifically, poorer skill learning was related to an attenuated shift in the E:I balance in the sulpiride condition, whereas this interaction was not evident in placebo. Our results demonstrate that exercise-primed motor skill acquisition is causally influenced by D2 receptor activity on motor cortical circuits.

The neutral Red probe location in large unilamellar vesicles: Influence of membrane composition and phase states

Biological membranes are complex and dynamic self-assembled structures composed of lipids and proteins. They depict emergent properties that are heterogeneous in the membrane plane. Therefore, their study is currently one of the greatest challenges in biophysics.The versatility of spectroscopic techniques has encouraged the development of new fluorescent probes capable of sensing different membrane properties. The Neutral Red (NR) molecule is a dye that protonates at pH < 6.8 (pKa = 6.8), leading to the charged species (NRH+). NR is widely employed in molecular biology to assess cell viability and mitochondrial function, and to sense intracellular pH. However, the photophysical behavior of NR in complex media such as membranes were not systematically characterized even though of the wide application in molecular biology.In this work, the photophysical behavior of NNR (pH 8 neutral specie) and NRH+ (pH 4) was studied by employing large unilamellar vesicles (LUVs) with different compositions, membrane phase states.The results of this study show that the spectroscopic behavior of NNR is more sensitive to the membrane composition than that of NRH+. The wavelength of the maximum emission of NNR was at shorter wavelengths in liquid-disordered (Ld) membranes compared to gel or liquid-ordered (Lo) membranes. The Red-edge excitation shifts (REES) and Anisotropy magnitudes were lower in membranes in the gel or Lo, compared to membranes in Ld. The same tendency was observed for NRH+, although the spectral changes in this case were smaller, as well as the changes in the REES effect, and anisotropy magnitudes were lower related to the NR species. We suggest that NNR and NRH+ are localized in different regions of the membrane depending on the membrane phase estate. NR and NRH+ are located in more internal regions of the interface in membranes in Ld phase state, whereas in gel or Lo phase membranes, NR is located in a more external region, and NRH+ is adsorbed on the membrane surface. Consequently, the spectral signals of both, NNR and NRH+, correspond to the species distributed in different locations of the membrane in Ld compared to gel and Lo phase. Therefore, the information provided by the probe inserted in different membranes is not directly comparable.

In-depth investigation of the effect of pH on the autofluorescence properties of DPF3b and DPF3a amyloid fibrils

Double PHD fingers 3 (DPF3) protein exists as two splicing variants, DPF3b and DPF3a, the involvement of which in human cancer and neurodegeneration is beginning to be increasingly recognised. Both isoforms have recently been identified as intrinsically disordered proteins able to undergo amyloid fibrillation. Upon their aggregation, DPF3 proteins exhibit an intrinsic fluorescence in the visible range, referred to as deep-blue autofluorescence (dbAF). Comprehension of such phenomenon remaining elusive, we investigated in the present study the influence of pH on the optical properties of DPF3b and DPF3a fibrils. By varying the excitation wavelength and the pH condition, the two isoforms were revealed to display several autofluorescence modes that were defined as violet, deep-blue, and blue-green according to their emission range. Complementarily, analysis of excitation spectra and red edge shift plots allowed to better decipher their photoselection mechanism and to highlight isoform-specific excitation-emission features. Furthermore, the observed violation to Kasha-Vavilov’s rule was attributed to red edge excitation shift effects, which were impacted by pH-mediated H-bond disruption, leading to changes in intramolecular charge and proton transfer, or π-electrons delocalisation. Finally, emergence of different autofluorescence emitters was likely related to structurally distinct fibrillar assemblies between isoforms, as well as to discrepancies in the amino acid composition of their aggregation prone regions.

Measurement of the Excitation Spectrum of a Dipolar Gas in the Macrodroplet Regime.

The excitation spectrum of a cigar-shaped strongly dipolar quantum gas at the crossover from a Bose-Einstein condensate to a trapped macrodroplet is predicted to exhibit peculiar features-a strong upward shift of low momentum excitation energies together with a strong multiband response for high momenta. By performing Bragg spectroscopy over a wide range of momenta, we observe both key elements and also confirm the predicted stiffening of excitation modes when approaching the macrodroplet regime. Our measurements are in good agreement with numerical calculations taking into account finite size effects.

Divergent changes in PBN excitability in a mouse model of neuropathic pain.

The transition from acute to chronic pain involves maladaptive plasticity in central nociceptive pathways. Growing evidence suggests that changes within the parabrachial nucleus (PBN), an important component of the spino-parabrachio-amygdaloid pain pathway, are key contributors to the development and maintenance of chronic pain. In animal models of chronic pain, PBN neurons become sensitive to normally innocuous stimuli and responses to noxious stimuli become amplified and more often produce after-discharges that outlast the stimulus. Using ex vivo slice electrophysiology and two mouse models of neuropathic pain, sciatic cuff and chronic constriction of the infraorbital nerve (CCI-ION), we find that changes in the firing properties of PBN neurons and a shift in inhibitory synaptic transmission may underlie this phenomenon. Compared to PBN neurons from shams, a larger proportion of PBN neurons from mice with a sciatic cuff were spontaneously active at rest, and these same neurons showed increased excitability relative to shams. In contrast, quiescent PBN neurons from cuff mice were less excitable than those from shams. Despite an increase in excitability in a subset of PBN neurons, the presence of after-discharges frequently observed in vivo were largely absent ex vivo in both injury models. However, GABAB-mediated presynaptic inhibition of GABAergic terminals is enhanced in PBN neurons after CCI-ION. These data suggest that the amplified activity of PBN neurons observed in rodent models of chronic pain arise through a combination of changes in firing properties and network excitability.Significance Statement Hyperactivity of neurons in the parabrachial nucleus (PBN) is causally linked to exaggerated pain behaviors in rodent models of chronic pain but the underlying mechanisms remain unknown. Using two mouse models of neuropathic pain, we show the intrinsic properties of PBN neurons are largely unaltered following injury. However, subsets of PBN neurons become more excitable and GABAB receptor mediated suppression of inhibitory terminals is enhanced after injury. Thus, shifts in network excitability may be a contributing factor in injury induced potentiation of PBN activity.

Understanding 2p core-level excitons of late transition metals by analysis of mixed-valence copper in a metal-organic framework.

The L2,3-edge X-ray absorption spectra of late transition metals such as Cu, Ag, and Au exhibit absorption onsets lower in energy for higher oxidation states, which is at odds with the measured spectra of earlier transition metals. Time-dependent density functional theory calculations for Cu2+/Cu+ reveal a larger 2p core-exciton binding energy for Cu2+, overshadowing shifts in single-particle excitation energies with respect to Cu+. We explore this phenomenon in a Cu+ metal-organic framework with ∼12% Cu2+ defects and find that corrections with self-consistent excited-state total energy differences provide accurate XAS peak alignment.

High-temperature effects for transition state calculations in solids.

Transition state calculation is a critical technique to understand and predict versatile dynamical phenomena in solids. However, the transition state results obtained at 0K are often utilized for the prediction or interpretation of dynamical processes at high temperatures, and the error bars of such an approximation are largely unknown. In this benchmark study, all the major temperature effects, including lattice expansion, lattice vibration, electron excitation, and band-edge shift, are evaluated with first-principles calculations for defect diffusion in solids. With the inclusion of these temperature effects, the notable discrepancies between theoretical predictions at 0K and the experimental diffusivities at high temperatures are dramatically reduced. In particular, we find that lattice expansion and lattice vibration are the dominant factors lowering the defect formation energies and hopping barriers at high temperatures, but the electron excitation exhibits minor effects. In sharp contrast to typical assumptions, the attempt frequency with lattice expansion and vibration varies significantly with materials: several THz for aluminum bulk but surprisingly over 500THz for 4H-SiC. For defects in semiconductors, the band-edge shift is also significant at high temperatures and plays a vital role in defect diffusion. We expect that this study would help accurately predict the dynamical processes at high temperatures.

The shift of excitation spectra at blue edge of emission (BEEmS) as a new methodology to probe heterogeneity

Many current methods for detecting molecular-level heterogeneity are complex and require stringent data analysis, limiting their widespread use. Herein, we propose a novel edge effect, i.e., the shift of excitation spectra at the blue edge of emission (which we termed as Blue Edge Emission Shift, BEEmS), to perceive the structural heterogeneity. This method is simple and can be easily implemented with commonly available fluorimeter. Red edge excitation shift (REES), a related technique, is already in use, but like most other known techniques, its usefulness is constrained by its dependence on environmental rigidity. Our method does not suffer from this drawback significantly. We showed the generality of the proposed method taking various chemically and biologically heterogenous systems including molecular liquid, deep eutectic solvents, organic cavitand, micelle and protein. BEEmS certainly comes out as a more effective sensor of heterogeneity than REES in certain cases (like denatured protein, hydrophobic deep eutectic solvent and SDS micelle) where solvation time is not sufficiently slow to be detected by REES, but can be measured though BEEmS. Furthermore, unlike most existing techniques, domain-specific heterogeneity of a model multi-domain protein is successfully measured.

Red edge excitation shift spectroscopy is highly sensitive to tryptophan composition.

Red edge excitation shift (REES) spectroscopy relies on the unique emission profiles of fluorophore-solvent interactions to profile protein molecular dynamics. Recently, we reported the use of REES to compare the stability of 32 polymorphic IgG antibodies natively containing tryptophan reporter fluorophores. Here, we expand on this work to investigate the sensitivity of REES to variations in tryptophan content using a subset of IgG3 antibodies containing arginine to tryptophan polymorphisms. Structural analysis revealed that the additional tryptophan residues were situated in highly solvated environments. Subsequently, REES showed clear differences in fluorescence emission profiles when compared with the unmutated variants, thereby limiting direct comparison of their structural dynamics. These findings highlight the exquisite sensitivity of REES to minor variations in protein structure and tryptophan composition.

Electrophysiological properties of layer 2/3 pyramidal neurons in the primary visual cortex of a retinitis pigmentosa mouse model (rd10).

Retinal degeneration is one of the main causes of visual impairment and blindness. One group of retinal degenerative diseases, leading to the loss of photoreceptors, is collectively termed retinitis pigmentosa. In this group of diseases, the remaining retina is largely spared from initial cell death making retinal ganglion cells an interesting target for vision restoration methods. However, it is unknown how downstream brain areas, in particular the visual cortex, are affected by the progression of blindness. Visual deprivation studies have shown dramatic changes in the electrophysiological properties of visual cortex neurons, but changes on a cellular level in retinitis pigmentosa have not been investigated yet. Therefore, we used the rd10 mouse model to perform patch-clamp recordings of pyramidal neurons in layer 2/3 of the primary visual cortex to screen for potential changes in electrophysiological properties resulting from retinal degeneration. Compared to wild-type C57BL/6 mice, we only found an increase in intrinsic excitability around the time point of maximal retinal degeneration. In addition, we saw an increase in the current amplitude of spontaneous putative inhibitory events after a longer progression of retinal degeneration. However, we did not observe a long-lasting shift in excitability after prolonged retinal degeneration. Together, our results provide evidence of an intact visual cortex with promising potential for future therapeutic strategies to restore vision.

Effect of Palmitic Acid on Tertiary Structure of Glycated Human Serum Albumin

Non-enzymatic glycation is a process, which can be best described as a significant posttranslational modification of various proteins. It emerges in hyperglycemic conditions and may have an impact on albumin stability as well as its activity and physical and chemical properties, essentially affecting all its physiological functions. The goal of this research was to answer the following questions: (i) how does the glycation of defatted human serum albumin by glucose–fructose syrup (GFS) alter its tertiary structure; (ii) does palmitic acid (PA), a component of palm oil, affect the in vitro glycation process and cause conformational changes of glycated albumin; and (iii) does PA inhibit the formation of Advanced Glycation End-Products (AGEs)? Therefore, in order to point out differences in the tertiary structure of macromolecules, the absorption and emission of fluorescence spectra and their second derivatives, excitation fluorescence and synchronous spectra, Red-Edge Excitation Shift (REES effect), and the degree of modification of sulfhydryl groups of defatted, non-glycated (HSA) and glycated human serum albumin (gHSA) with GFS and glycated with GFS and PA were investigated. In the present study, it has been confirmed that the glycation of albumin in the presence of GFS and PA causes changes in both HSA and gHSA tertiary structures, respectively. Moreover, palmitic acid, at ratios of 1.5:1 and 3:1 with glycated albumin, does not exhibit inhibition of AGEs formation. This study indicates the fact that the structural changes, especially those of glycated albumin, are important for treatment planning because the type of the interaction between the components and their primary transporter may be altered as the disease progresses or in the elderly.

Binding characteristics and conformational changes in alpha-2-macroglobulin by the dietary flavanone naringenin: biophysical and computational approach

In the present study, we investigated the interaction of alpha-2-macroglobulin (α2M) with naringenin using multi-spectroscopic, molecular docking, and molecular simulation approaches to identify the functional changes and structural variations in the α2M structure. Our study suggests that naringenin compromised α2M anti-proteinase activity. The results of absorption spectroscopy and fluorescence measurement showed that naringenin-α2M formed a complex with a binding constant of (kb)∼104, indicative of moderate binding. The value of ΔG° in the binding indicates the process to be spontaneous and the major force responsible to be hydrophobic interaction. The findings of FRET reveal the binding distance between naringenin and the amino acids of α2M was 2.82 nm. The secondary structural analysis of α2M with naringenin using multi-spectroscopic methods like synchronous fluorescence, red-edge excitation shift (REES), FTIR, and CD spectra further confirmed the significant conformational alterations in the protein. Molecular docking approach reveals the interactions between naringenin and α2M to be hydrogen bonds, van der Waals forces, and pi interactions, which considerably favour and stabilise the binding. Molecular dynamics modelling simulations also supported the steady binding with the least RMSD deviations. Our study suggests that naringenin interacts with α2M to alter its confirmation and compromise its activity. Communicated by Ramaswamy H. Sarma

High‐Performance and Humidity‐Resistant Direct‐Current Triboelectric Nanogenerator by Coupling Method Combing Charge Excitation and Phase Shift

AbstractTriboelectric nanogenerator (TENG)’s use is constrained by their surface charge density and alternating current output. This study therefore proposes a coupling output method combining charge excitation and phase shift to realize an almost constant high‐performance direct‐current (DC) and DC‐TENG is designed to harvest the energy of flowing water. The output characteristic of the DC‐TENG with or without charge excitation is thoroughly investigated in the experiment. The impact on the effectiveness of phases (P) and groups (G) is further proven by comparing and quantifying the current wave. The operation capability in a water environment is further demonstrated by the transmitted charge's attenuation of roughly 10.84% after the environment's humidity is increased to 90% RH. According to the findings of the investigation, DC‐TENG with 3P4G can reach almost constant DC with the root mean square value of 29.1 μA and the crest factor of 1.23. And the 220 μF capacitor can be charged to 5 V within 63 s and the peak output power of 9.18 mW is obtained. Finally, an experimental platform is established to simulate the water environment and the thermography is steadily powered. This scheme has reference significance to realize high‐performance DC output in the water environment.

Thermodynamic insight into the interaction mechanism of an ESIPT drug with a serum transport protein: Slower solvent-relaxation dynamics explored through wavelength-sensitive fluorescence behavior

Recently, 3,5-diiodosalicylic acid (3,5-DISA) has garnered enormous research attention owing to its wide-spread medicinal/biological applications, use as a model to study the role of halogen bonding in drug-protein interaction and so forth. However, the arena of the study of interaction of 3,5-DISA with relevant biological targets, particularly serum transport protein still starves for meticulous exploration of a number of fundamental aspects, such as, the thermodynamics and strength of binding, effect of drug binding on the native protein conformation and functionality, rotational and solvent-relaxation dynamics within the protein scaffolds etc. The present investigation endeavors to unveil these important aspects of 3,5-DISA:HSA interaction scenario. Our spectroscopic results demonstrate a remarkable modification of the excited-state intramolecular proton transfer (ESIPT) photophysics of 3,5-DISA upon interaction with HSA. A detailed isothermal titration calorimetry (ITC) study reveals that the interaction process is governed by favorable enthalpic (∆H0 < 0) and unfavorable entropic (T∆S0 < 0) contributions. The modulation of the hydration structure at the interfaces accompanying the binding phenomenon is also delineated in this context. Circular dichroism (CD) spectroscopy has been exploited to show the slight perturbation of the native protein conformation as a result of drug binding. In complementarity, the functionality of HSA (in terms of esterase-like activity) has also been shown to decrease with added 3,5-DISA. Our cumulative exploration of the wavelength-sensitive fluorescence parameters including red-edge excitation shift (REES) points out a remarkably slower rate of solvent-relaxation dynamics of the HSA-bound 3,5-DISA in the photoexcited state. The modification of the rotational-relaxation behavior of 3,5-DISA within the protein is also addressed and rationalized on the basis of the two-step and wobbling-in-cone model.

Role of dynamic Stark shifts in strong-field excitation and subsequent ionization

Role of dynamic Stark shifts in strong-field excitation and subsequent ionization

HIGH ANTIOXIDATION-CAPACITY STEM CELLS ENHANCE CARTILAGE REGENERATION

Stem cells are known to have low levels of intracellular reactive oxygen species (ROS) and high levels of glutathione. ROS are thought to interact with several pathways that affect the transcription machinery required for stem cell differentiation, and are critical for maintaining stem cell function. In this study, we are developing a new fluorescent probe that rapidly and reversibly reacts with glutathione (GSH), the most abundant non-protein thiol in living cells that acts as an antioxidant and redox regulator.Multipotent perivascular progenitor cells derived from human ESCs (hESC-PVPCs): Differentiated ESCs as embryoid bodies in the presence of BMP4 to induce mesoderm differentiation followed by a simple cell selection strategy using attachment of single cells onto collagen-coated dishes. Differential gene expression profiling was performed among H9 hESCs, EBs induced by BMP4 and naturally selected CD140B+CD44+ population at Day 7 (PVPCs). Colony-forming assay: GSHhigh and GSHlow PVPCs were plated on 10-cm tissue culture-treated polystyrene dishes in triplicate in growth medium and cultured for 14 days. Transwell migration assay: GSHhigh and GSHlow PVPCs at passage 4 were resuspended at 1 × 106/mL in the migration medium and seeded in the upper chamber. The following human recombinant SDF-1 and PDGF-AA proteins were used as chemoattractants in the lower compartment.Probe-GSH conjugate shows shifts in fluorescence excitation and emission spectra that enables ratiometric measurement of GSH levels. Using these properties, stem cells can be purified by FACS-based technology according to intracellular GSH level. We are developing a protocol both for comparing GSH level in stem cell from different culture conditions and for preparing stem cells with high-GSH level . Our results reveal that GSHhigh PVPC purified by FACS show increased colony forming ability compared with that GSHlow PVPC, indicating that intracellular GSH contributes to the maintenance of stemness. Moreover, transplantation of GSHlow PVPC is more effective than that of GSHlow PVPC for cartilage regeneration in osteochondral defect.This technique enable FACS-based sorting of stem cells according to intracellular GSH levels and thus investigation of functional role of GSH (high antioxidant capacity) in the stem cell maintenance and chondrogenic differentiation.