PKH26 labeling of extracellular vesicles: Characterization and cellular internalization of contaminating PKH26 nanoparticles

PKH lipophilic dyes are highly fluorescent and stain membranes by intercalating their aliphatic portion into the exposed lipid bilayer. They have established use in labeling and tracking of cells in vivo and in vitro. Despite wide use of PKH-labeled extracellular vesicles (EVs) in cell targeting and functional studies, nonEV-associated fluorescent structures have never been examined systematically, nor was their internalization by cells. Here, we have characterized PKH26-positive particles in lymphoblastoid B exosome samples and exosome-free controls stained by ultracentrifugation, filtration, and sucrose-cushion-based and sucrose-gradient-based procedures, using confocal imaging and asymmetric-flow field-flow fractionation coupled to multi-angle light-scattering detector analysis. We show for the first time that numerous PKH26 nanoparticles (nine out of ten PKH26-positive particles) are formed during ultracentrifugation-based exosome staining, which are almost indistinguishable from PKH26-labeled exosomes in terms of size, surface area, and fluorescence intensity. When PKH26-labeled exosomes were purified through sucrose, PKH26 nanoparticles were differentiated from PKH26-labeled exosomes based on their reduced size. However, PKH26 nanoparticles were only physically removed from PKH26-labeled exosomes when separated on a sucrose gradient, and at the expense of low PKH26-labeled exosome recovery. Overall, low PKH26-positive particle recovery is characteristic of filtration-based exosome staining. Importantly, PKH26 nanoparticles are internalized by primary astrocytes into similar subcellular compartments as PKH26-labeled exosomes. Altogether, PKH26 nanoparticles can result in false-positive signals for stained EVs that can compromise the interpretation of EV internalization. Thus, for use in EV uptake and functional studies, sucrose-gradient-based isolation should be the method of choice to obtain PKH26-labeled exosomes devoid of PKH26 nanoparticles.     

Characterization and efficacy of PKH26 as a probe to study the replication history of the human hematopoietic KG1a progenitor cell line

The PKH26 dye can, in principle, be used for the study of asymmetric cell divisions (ASDs). A requirement for the identification of ASDs based on fluorescence intensity is that the PKH26 dye is distributed equally between daughter cells at each division, but this has not been demonstrated at a single-cell level. The efficacy of PKH26 as a probe for the study of ASDs was examined using the human hematopoietic KG1a cell. An automated time-lapse fluorescent microscope system was used to determine changes in cell size and fluorescence intensity during culture, and track cell divisions. The images of daughter cells were analyzed using the Isee software to determine the distribution of PKH26 dye between daughter cells. Ratios of cell size, mean fluorescence intensity, and total fluorescence intensity were calculated by dividing the values for one daughter cell by the value of the other daughter cell. The ratios for cell size, mean intensity, and total intensity were 1.13 +/- 0.12, 1.08 +/- 0.07, and 1.15 +/- 0.14 (mean +/- SD), respectively. Thus, PKH26 is not distributed equally to both daughter cells upon cell division. However, the replication history of individual KG1a cells can be reliably deduced for up to three divisions based solely on the mean and total fluorescence intensity of the PKH26 dye, using PKH26 concentrations below the chemical and phototoxic limits (2 microM).     

Innocuousness and intracellular distribution of PKH67: A fluorescent probe for cell proliferation assessment

PKH dyes were initially developed by Horan et al. to provide appropriate probes for in vitro and in vivo cell tracking. It has been reported for many cell types that PKH bind irreversibly to the cell membrane without significantly affecting cell growth. Thus, these probes provide an opportunity for long-term cell monitoring and the identification of cells of interest among a heterogeneous cell population. An important feature is that upon cell division, the probe is partitioned equally between each daughter cell, making it possible to quantify tell fluorescence by flow cytometry. In this situation. the flow cytometric study of PKH67 characteristics shows that this probe does not affect the main cell-functions such as viability or proliferation. Moreover, the intracellular distribution of PKH67 is demonstrated by following its kinetics of internalization by confocal microscopy. These results present PKH67 as a probe suitable for dynamic analysis of cell proliferation as well as the study of intracellular localization and membrane recycling mechanisms.     

PKH26 Staining Defines Distinct Subsets of Normal Human Colon Epithelial Cells at Different Maturation Stages

BACKGROUND AND AIM: Colon crypts are characterized by a hierarchy of cells distributed along the crypt axis. Aim of this paper was to develop an in vitro system for separation of epithelial cell subsets in different maturation stages from normal human colon. METHODOLOGY AND MAJOR FINDINGS: Dissociated colonic epithelial cells were stained with PKH26, which allows identification of distinct populations based on their proliferation rate, and cultured in vitro in the absence of serum. The cytofluorimetric expression of CK20, Msi-1 and Lgr5 was studied. The mRNA levels of several stemness-associated genes were also compared in cultured cell populations and in three colon crypt populations isolated by microdissection. A PKH(pos) population survived in culture and formed spheroids; this population included subsets with slow (PKH(high)) and rapid (PKH(low)) replicative rates. Molecular analysis revealed higher mRNA levels of both Msi-1 and Lgr-5 in PKH(high) cells; by cytofluorimetric analysis, Msi-1(+)/Lgr5(+) cells were only found within PKH(high) cells, whereas Msi-1(+)/Lgr5(-) cells were also observed in the PKH(low) population. As judged by qRT-PCR analysis, the expression of several stemness-associated markers (Bmi-1, EphB2, EpCAM, ALDH1) was highly enriched in Msi-1(+)/Lgr5(+) cells. While CK20 expression was mainly found in PKH(low) and PKH(neg) cells, a small PKH(high) subset co-expressed both CK20 and Msi-1, but not Lgr5; cells with these properties also expressed Mucin, and could be identified in vivo in colon crypts. These results mirrored those found in cells isolated from different crypt portions by microdissection, and based on proliferation rates and marker expression they allowed to define several subsets at different maturation stages: PKH(high)/Lgr5(+)/Msi-1(+)/CK20(-), PKH(high)/Lgr5(-)/Msi-1(+)/CK20(+), PKH(low)/Lgr5(-)/Msi-1(+)/Ck20(-), and PKH(low)/Lgr5(-)/Msi-1(-)/CK20(+) cells. CONCLUSIONS: Our data show the possibility of deriving in vitro, without any selection strategy, several distinct cell subsets of human colon epithelial cells, which recapitulate the phenotypic and molecular profile of cells in a discrete crypt location.     

Biologic properties of gadolinium diethylenetriaminepentaacetic acid-labeled and PKH26-labeled human umbilical cord mesenchymal stromal cells

Background aimsThis study was conducted to characterize gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA)-labeled and PKH26-labeled human umbilical cord mesenchymal stromal cells (HuMSCs) and to track them with magnetic resonance imaging (MRI) in vitro and in vivo.MethodsHuMSCs were isolated from umbilical cords and expanded in vitro. Cells were sequentially labeled with Gd-DTPA and PKH26. The labeling efficiency was determined by spectrophotometry measurements, and the longevity of Gd-DTPA maintenance was measured with MRI. The influence of double labeling on cellular biologic properties was assessed by cell proliferation, viability, differentiation, cycle and apoptosis. Transplantation of double-labeled HuMSCs or placebo was performed in 39 female Sprague-Dawley rats. Leak point pressure and maximal bladder capacity were measured in animals 6 weeks after injection.ResultsThe T1 values and signal intensity on T1-weighted imaging of labeled cells were significantly higher than the control group (P < 0.05). The signal intensity on T1-weighted imaging of labeled cells was retained >14 days in vitro and in vivo. There was no significant difference in the cell cycle, cell apoptosis, cell proliferation and cell viability between labeled and unlabeled HuMSCs (P > 0.05). After double labeling, HuMSCs were still capable of differentiating into osteoblasts and adipocytes. Periurethrally injected HuMSCs in the rats significantly improved leak point pressure and maximal bladder capacity.ConclusionsHuMSCs were successfully labeled with Gd-DTPA and PKH26. This labeling method is reliable and efficient and can be applied for tracking cells in vitro and in vivo without altering cellular biologic properties.     

PKH26 as a fluorescent label for live human umbilical mesenchymal stem cells

To determine whether PKH26 labeling affects the morphologies, phenotypes, proliferation, and secretion abilities of human umbilical mesenchymal stromal cells (HUMSCs) were investigated. Isolated HUMSCs were labeled with PKH26, and cell morphology was observed under microscope. Cell cycle, apoptotic cell death, expression of PKH26, and the proliferation rate were evaluated. Additionally, fluorescence intensity of PKH26 labeling at different passage times was quantified. There were no detectable differences in cell morphology, cell growth, and proliferation rate after PKH26 labeling. In addition, fluorescence intensity of PKH26 labeling was gradually reduced with increase of the passage times. The PKH26 labeling disappeared after passage six times. In summary, PKH26 labeling is a safe and effective way to label live HUMSCs.     

Fluorescent dyes for lymphocyte migration and proliferation studies

Fluorescent dyes are increasingly being exploited to track lymphocyte migration and proliferation. The present paper reviews the properties and performance of some 14 different fluorescent dyes that have been used during the last 20 years to monitor lymphocyte migration. Of the 14 dyes discussed, two stand out as being the most versatile in terms of long-term tracking of lymphocytes and their ability to quantify lymphocyte proliferation. They are the intracellular covalent coupling dye carboxyfluorescein diacetate succinimidyl ester (CFSE) and the membrane inserting dye PKH26. Both dyes have the advantage that they can be used to track cell division, both in vitro and in vivo, due to the progressive halving of the fluorescence intensity of the dyes in cells after each division. However, CFSE appears to have the edge over PKH26 based on homogeneity of lymphocyte staining and cost. Two other fluorescent dyes, although not suitable for lymphocyte proliferation studies, are valuable tracking dyes for short-term (up to 3 day) lymphocyte migration experiments, namely the DNA-binding dye Hoechst 33342 and the cytoplasmic dye calcein. In the future it is highly likely that additional fluorescent dyes, with different spectral properties to CFSE, will become available, as well as membrane inserting fluorescent dyes that more homogeneously label lymphocytes than PKH26.     

Studies on curcumin and curcuminoids. XXIX. Photoinduced cytotoxicity of curcumin in selected aqueous preparations.

Natural curcumin was evaluated as a potential photosensitizer for oral applications. The photocytotoxicity of curcumin on salivary gland acinar cells (SM 10-12) was investigated in five aqueous preparations consisting of 5% DMSO, non-ionic micelles, cyclodextrin, liposomes, or a hydrophilic polymer. The difference in phototoxic effects between natural curcumin and synthetic curcumin was examined. Cytotoxicity in SM 10-12 cells exposed to curcumin in the concentration range 0.4-13.5 microM was investigated by MTT test, a fluorescence-staining microscopic test, and by Western immunoblotting techniques. The potential formation of a photoreaction product, hydrogen peroxide, was evaluated by a fluorescence assay. The light source was a halogen lamp used in the dental clinic, emitting mainly in the blue part of the spectrum. The phototoxic effect on SM 10-12 cells was dependent on curcumin concentration, the light dose and the type of preparation. Natural and synthetic curcumin induced phototoxicity to the same extent. Significant effects on the cells were obtained at low curcumin concentrations (< or =0.5 microM) and at a low light dose (< or =6 J cm(-2)), after 3 h incubation. Neither the activation of caspases-3, -7, -8 or -9, nor the formation of hydrogen peroxide could be detected in cells exposed to curcumin and light. The liposome preparation was the most efficient vehicle for curcumin to induce cell death. The phototoxic effect induced by curcumin is highly dependent on the type of preparation. Curcumin might be a potential photosensitizer in the treatment of oral lesions and cancers provided careful selection of the vehicle.     

Concurrent measurement of the survival of two populations of rabbit platelets labeled with either two PKH lipophilic dyes or two concentrations of biotin

BACKGROUND: To avoid radioisotopic labeling and permit comparison of the survival of two platelet populations concurrently in one animal, we compared simultaneous recoveries and survival times of homologous rabbit platelets labeled in vitro with the lipophilic dyes PKH26 (red fluorescing) and PKH67 (green fluorescing) and with two levels of biotin (low, 1 microg/ml; high, 10 microg/ml). METHODS: Blood samples were drawn up to 96 h postinfusion and analyzed by flow cytometry. Biotin-labeled samples were incubated with phycoerythrin-streptavidin before analysis. RESULTS: Recovery of PKH26-labeled platelets at 1 h was lower (37.5%) than that of PKH67-labeled platelets (47.3%; P < 0.001). Platelet survival times were 62.4 and 61.9 h. Recoveries at 1 h of platelets labeled with two levels of biotin were similar (86.6% and 84.6%) and greater than those of PKH-labeled platelets (P < 0.001). Survival of platelets labeled with biotin did not differ (low, 83.3 h; high, 85.2 h) and was longer than for PKH-labeled platelets (P < 0.01). Labeling methods did not activate platelets (measured by P-selectin expression), nor did they affect platelet responses to adenosine diphosphate (ADP), collagen, or thrombin. CONCLUSIONS: Labeling with two levels of biotin is superior to labeling with PKH dyes, and is useful for measuring concurrently the survival of two differing platelet populations.     

PKH26标记的人羊膜间充质干细胞在宫腔粘连大鼠中的迁移示踪

为了观察PKH26标记的人羊膜间充质干细胞(hAMSCs)在宫腔粘连大鼠子宫内膜中的迁移情况,文中提取鉴定及PKH26标记hAMSCs,检测PKH26染色剂对hAMSCs生物学特性的影响;利用机械感染双重法建立大鼠宫腔粘连模型并经尾静脉移植PKH26标记的hAMSCs,荧光共聚焦显微镜下观察PKH26标记的hAMSCs移植后在大鼠子宫内膜中的分布情况。结果显示,PKH26染色剂对细胞的活性、周期、凋亡等无明显影响,PKH26标记的阳性细胞主要分布在大鼠受损的子宫内膜中。表明PKH26标记技术是一种安全有效的示踪方法,可用于hAMSCs移植在治疗宫腔粘连时的示踪研究。     

Phototoxic Action Spectrum on a Retinal Pigment Epithelium Model of Age-Related Macular Degeneration Exposed to Sunlight Normalized Conditions

Among the identified risk factors of age-related macular degeneration, sunlight is known to induce cumulative damage to the retina. A photosensitive derivative of the visual pigment, N-retinylidene-N-retinylethanolamine (A2E), may be involved in this phototoxicity. The high energy visible light between 380 nm and 500 nm (blue light) is incriminated. Our aim was to define the most toxic wavelengths in the blue-green range on an in vitro model of the disease. Primary cultures of porcine retinal pigment epithelium cells were incubated for 6 hours with different A2E concentrations and exposed for 18 hours to 10 nm illumination bands centered from 380 to 520 nm in 10 nm increments. Light irradiances were normalized with respect to the natural sunlight reaching the retina. Six hours after light exposure, cell viability, necrosis and apoptosis were assessed using the Apotox-Glo Triplex™ assay. Retinal pigment epithelium cells incubated with A2E displayed fluorescent bodies within the cytoplasm. Their absorption and emission spectra were similar to those of A2E. Exposure to 10 nm illumination bands induced a loss in cell viability with a dose dependence upon A2E concentrations. Irrespective of A2E concentration, the loss of cell viability was maximal for wavelengths from 415 to 455 nm. Cell viability decrease was correlated to an increase in cell apoptosis indicated by caspase-3/7 activities in the same spectral range. No light-elicited necrosis was measured as compared to control cells maintained in darkness. Our results defined the precise spectrum of light retinal toxicity in physiological irradiance conditions on an in vitro model of age-related macular degeneration. Surprisingly, a narrow bandwidth in blue light generated the greatest phototoxic risk to retinal pigment epithelium cells. This phototoxic spectrum may be advantageously valued in designing selective photoprotection ophthalmic filters, without disrupting essential visual and non-visual functions of the eye.     

The phototoxicity of phenothiazinium derivatives against Escherichia coli and Staphylococcus aureus

Phenothiazinium dyes, and derivatives, were tested for toxicity to Escherichia coli and Staphylococcus aureus. The dyes were generally lipophilic (log P>1) and showed inherent dark toxicity (minimum lethal concentrations: 3.1-1000 microM). Dye illumination (total light dose of 3.15 J cm(-1) over 30 min) led to up to eight-fold reductions in minimum lethal concentrations. Most of the illuminated dyes showed significant relative singlet oxygen yields (phi'delta: 0.18-1.35) suggesting a type II mechanism of generating a phototoxic response. Although generally up to six-fold more effective against S. aureus, the dyes tested efficiently killed E. coli and may be of particular use in combating Gram-negative pathogens.     

Reliable Screening of Dye Phototoxicity by Using a Caenorhabditis elegans Fast Bioassay

Phototoxicity consists in the capability of certain innocuous molecules to become toxic when subjected to suitable illumination. In order to discover new photoactive drugs or characterize phototoxic pollutants, it would be advantageous to use simple biological tests of phototoxicy. In this work, we present a pilot screening of 37 dyes to test for phototoxic effects in the roundworm Caenorhabditis elegans. Populations of this nematode were treated with different dyes, and subsequently exposed to 30 min of white light. Behavioral outcomes were quantified by recording the global motility using an infrared tracking device (WMicrotracker). Of the tested compounds, 17 dyes were classified as photoactive, being phloxine B, primuline, eosin Y, acridine orange and rose Bengal the most phototoxic. To assess photoactivity after uptake, compounds were retested after washing them out of the medium before light irradiation. Dye uptake into the worms was also analyzed by staining or fluorescence. All the positive drugs were incorporated by animals and produced phototoxic effects after washing. We also tested the stress response being triggered by the treatments through reporter strains. Endoplasmic reticulum stress response (hsp-4::GFP strain) was activated by 22% of phototoxic dyes, and mitochondrial stress response (hsp-6::GFP strain) was induced by 16% of phototoxic dyes. These results point to a phototoxic perturbation of the protein functionality and an oxidative stress similar to that reported in cell cultures. Our work shows for the first time the feasibility of C. elegans for running phototoxic screenings and underscores its application on photoactive drugs and environmental pollutants assessment.     

Laser scanning confocal microscopy of the hearing organ: fluorochrome-dependent cellular damage is seen after overexposure

In order to combine laser confocal microscopy with physiological measurements, a number of conditions have to be met: the dye must not be toxic to the cells the laser light itself must not damage the cells; and the excitation of the fluorochrome during imaging must not generate products with toxic effects. We have investigated these conditions the hearing organ of the guinea pig. Two dyes were used, namely, calcein-AM, which is metabolized in vital cells to a fluorescent product in the cytoplasm, and a lipophilic membrane dye. The effect of the dyes on cell function was tested in the intact hearing organ, maintained in the isolated temporal bone, by measuring the electrophysiological potentials generated by the sensory cells in response to tone pulses. The loading of the cells with the dyes had no adverse effects. The effect of the laser beam was explored on isolated coils from the cochlea. In two preparations, the specimens viewed in the confocal system were fixed and processed for electron microscopy. Identified cells were followed before, during, and after laser exposure and could ultimately be examined at the ultrastructural level. Exposure to the laser beam did not cause damage in unstained cells, even at high intensities. In stained tissue, confocal microscopy could safely be performed at normal beam intensity without causing ultrastructural changes. At high intensities, about 100 times normal for 60 times as long, irradiation damage was seen that was selective in that the cells stained with the different dyes exhibited damage at the different sites corresponding to the subcellular location of the dyes. Cells stained with calcein showed lysis of mitochondria and loss of cytoplasmic matrix, whereas cells stained with the styryl membrane dye showed swelling of subsurface cisternae, contortion of the cell wall, and shrinkage. The styryl dyes, in particular, which selectively stain the sensory and neuronal cells in the organ of Corti, could be exploited for phototoxic use.     

Metabolic toxicity of fluorescent stains on thawed cryopreserved bovine sperm cells

Several fluorescent probes, including derivatives of carboxyfluorescein, carbocyanine, ethidium, and rhodamine, have been used to assess sperm viability. However, the effects of these fluorescent dyes on the metabolic activity of sperm cells have not been systematically examined. This study was conducted to determine the effect of specific fluorescent stains on the metabolic processes of sperm. Cryopreserved bovine sperm cells were thawed, fluorescently stained, and examined using metabolic and flow cytometric techniques. Sperm were stained with either rhodamine 123 (Rhod-123), the aliphatic cell-tracking compound PKH2-GL, dihydro-ethidium (HED), the bisbenzimide stain Hoechst 33342 (Ho33342), or left unstained. The stained samples were compared for metabolic activity, cell staining pattern, and fluorescent intensity over a 180-min period. Samples stained with HED, Ho33342, and PKH2-GL had less oxygen uptake when compared with the unstained sperm samples (p greater than 0.05). Unstained samples and samples stained with Rhod-123 had similar oxygen consumption. The carbon dioxide produced during the 180 min was not different between controls and stained samples. Therefore, some fluorescent probes inhibit the oxygen metabolism of thawed, cryopreserved bovine sperm cells.     

Bone marrow and adipose stem cells can be tracked with PKH26 until post staining passage 6 in in vitro and in vivo

Tracking of transplanted cells has become an important procedure in cell therapy. We studied the in vitro dye retention, survival and in vivo tracking of stem cells with PKH26 dye. Sheep BMSCs and ADSCs were labeled with 2, 4 and 8 μmol of PKH26 and monitored for six passages. Labeled BMSCs and ADSCs acquired mean cumulative population doubling of 12.7 ± 0.4 and 14.6 ± 0.5; unlabeled samples had 13.8 ± 0.5 and 15.4 ± 0.6 respectively. Upon staining with 2, 4 and 8 μmol PKH26, BMSCs had retentions of 40.0 ± 5.8, 60.0 ± 2.9 and 95.0 ± 2.9%, while ADSCs had 92.0 ± 1.2, 95.0 ± 1.2 and 98.0 ± 1.2%. ADSCs retentions were significantly higher at 2 and 4 μmol. On dye retention comparison at 8 μmol and 4 μmol for BMSCs and ADSCs; ADSCs were significantly higher at passages 2 and 3. The viability of BMSCs reduced from 94.0 ± 1.2% to 90.0 ± 0.6% and ADSCs from 94.0 ± 1.2% to 52.0 ± 1.2% (p < 0.05) after 24 h. BMSCs had significant up regulation of the cartilage genes for both the labeled and the unlabeled samples compared to ADSCs (p < 0.05). PKH26 fluorescence was detected on the resected portions of the regenerated neo-cartilage. The recommended concentration of PKH26 for ADSCs is 2 μmol and BMSCs is 8 μmol, and they can be tracked up to 49 days.     

Evaluation of chemically-induced phototoxicity to aquatic organism using Paramecium as a model

Phototoxicity evaluation using Paramecium aurelia as a model revealed that 4 out of 21 pesticides produced lethal toxicity to cells. Four commonly used synthetic dyes (bromophenol blue, rose bengal, benzanthrone and methylene blue) also exhibited toxicity. Well known phototoxic agents like hematoporphyrin, riboflavin, and anthracene produced positive phototoxic response. Psoralen, a DNA cross-linking agent, also produced phototoxicity to the cells. The results clearly demonstrate that the synergistic action of chemical agents and sunlight produce lethal effects to aquatic organism.     

Photoinactivation effects of hematoporphyrin monomethyl ether on Gram-positive and -negative bacteria detected by atomic force microscopy

The photodynamic antimicrobial chemotherapy as a promising approach for efficiently killing pathogenic microbes is attracting increasing interest. In this study, the cytotoxic and phototoxic effects of hematoporphyrin monomethyl ether (HMME) on the Gram-positive and Gram-negative bacteria were investigated. The cell viability was assessed by colony-forming unit method, and the results indicated that there was no significant cytotoxicity but high phototoxicity in the examined concentrations. Notably, the Gram-positive bacteria were more sensitive to HMME in phototoxicity. Simultaneously, an atomic force microscope (AFM) was used to detect the changes in morphological and nanomechanical properties of bacteria before and after HMME treatment. AFM images indicate that upon photoinactivation, the bacterial surface changed from a smooth, homogeneous architecture to a heterogenous, crackled morphology. The force spectroscopy measurements reveal that the cell wall became less rigid and the Young’s modulus decreased about 50%, whereas the tip-cell-surface adhesion forces increased significantly compared to those of native cells. It was speculated that the photodynamic effects of HMME induced the changes in the chemical composition of the outer membrane and exposure of some proteins inside the envelope. AFM can be utilized as a powerful and sensitive method for studying the interaction between bacteria and drugs.     

Fluorescence cell imaging and manipulation using conventional halogen lamp microscopy

Technologies for vitally labeling cells with fluorescent dyes have advanced remarkably. However, to excite fluorescent dyes currently requires powerful illumination, which can cause phototoxic damage to the cells and increases the cost of microscopy. We have developed a filter system to excite fluorescent dyes using a conventional transmission microscope equipped with a halogen lamp. This method allows us to observe previously invisible cell organelles, such as the metaphase spindle of oocytes, without causing phototoxicity. Cells remain healthy even after intensive manipulation under fluorescence observation, such as during bovine, porcine and mouse somatic cell cloning using nuclear transfer. This method does not require expensive epifluorescence equipment and so could help to reduce the science gap between developed and developing countries.     

Unexpected durability of PKH 26 staining on rat adipocytes

In a case of autologous mature fat cell transplantation to an individual rat, persistence of adipose staining with PKH26 was detected 14.5 months later. Fluorescent fat cells with spotty surface markings were easily visible. Such a long period of persistence and stability of staining exceeds the expectation from previous reports, and the given half-life of the substance. This finding encourages the use of the dye for long-term follow-up of connective tissue cells, especially adipocytes and preadipocytes, following transplantation.