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Staining Properties of Oil Red O and a Method of Partial Purification of the Commercial Product
Abstract
1. A yellow-brown component of commercial oil red O separated by our technic of paper chromatography is shown to stain human serum proteins, particularly those that are coagulated by heat. The patterns of serum electropherograms obtained by coloring with the crude oil red O solution consist of a red lipid pattern superimposed on a brownish protein pattern.
2. A method is described for partial removal of the nonred components from the crude oil red O. Satisfactory coloration of lipoproteins was obtained with the "purified" oil red O.
3. It is demonstrated that areas of paper strip covered with protein may stain lighter with some oil red O fractions than the background filter paper.
... Morphologically, nascent lipid droplets initially appear as a perinuclear halo (Boone et al., 2000a), which will later coalesce into larger lipid droplets (Boone et al., 2000a). Ultimately, the cell contains a peripherally located nucleus and a unilocular lipid droplet (Russell and Ho, 1976), which can be stained with oil red O (Kutt and Tsaltas, 1959;Kinkel et al., 2004). The specific physiological transitions of adipofibroblasts through the adipogenesis differentiation program are unknown. ...
... After 12 d , the cells were fixed and stained (Kutt and Tsaltas, 1959;Kinkel et al., 2004), and the cell number and percent differentiation were determined. and Animal Health for evaluation of cortisol, insulin, glucose, and insulin-like growth factor -1 (IGF-1) levels, and fatty acid profiles. ...
... The adipofibroblast cultures were stained for lipid content as previously described (Kutt and Tsaltas, 1959;Kinkel et al., 2004). Briefly, the cultures were rinsed in phosphate buffered saline (PBS) to remove medium components and fixed in 10 % formalin for 10 min. ...
... Oil red O is a non-polar dye that stains triglycerides, lipids and some lipoproteins. Although not specific for adipocytes, it is frequently used to visualise intracellular lipid accumulation (KUTT and TSALTAS 1959;HAUSMAN 1981;KOOPMAN, SCHAART et al. 2001). ...
... Cells were fixated using 4 % neutral buffered paraformaldehyde (Apoteket, Stockholm, Sweden). All routine stainings were performed using previously described protocols (KUTT and TSALTAS 1959;LEV and SPICER 1964;MASON 1971;BILLS, EISENBERG et al. 1974;HAUSMAN 1981;WOODS, KHAN et al. 2007). Oil red O staining in paper I was quantified by examining randomly selected high power fields and counting stained and unstained cells. ...
... Lipid droplets (LDs) were stained with Oil Red O (Kutt & Tsaltas 1959 ). A stock of Oil Red O solution was prepared in 2-propanol (0.3%), vortexed and filtered through a 0.8 μm sieve before staining. ...
... The fold change induction by petroselinic acid after CLTC transient silencing was significantly reduced from 3.48 to 2.06 fold in the MALME-3M cells and from 3.15 to 2.15 in the MCF-7 cells. To corroborate the inhibitory effect of CLTC suppression on the accumulation of LDs, we employed the Oil Red O dye that specifically binds to neutral lipids (Kutt & Tsaltas 1959). After transfection and a 24 hour incubation period with petroselinic acid (100 μg/ml), MALME-3M and MCF-7 cells were stained with Oil Red O and hematoxylin (Figure 5E and 5F). ...
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Enforced cell transdifferentiation of human cancer cells is a promising alternative to conventional chemotherapy. We previously identified albumin-associated lipid- and, more specifically, saturated fatty acid-induced transdifferentiation programs in human cancer cells (HCCLs). In this study, we further characterized the adipocyte-like cells, resulting from the transdifferentiation of human cancer cell lines MCF-7 and MALME-3M, and proposed a common mechanistic approach for these transdifferentiating programs. We showed the loss of pigmentation in MALME-3M cells treated with albumin-associated lipids, based on electron microscopic analysis, and the overexpression of perilipin 2 (PLIN2) by western blotting in MALME-3M and MCF-7 cells treated with unsaturated fatty acids. Comparing the gene expression profiles of naive melanoma MALME-3M cells and albumin-associated lipid-treated cells, based on RNA sequencing, we confirmed the transcriptional upregulation of some key adipogenic gene markers and also an alternative splicing of the adipogenic master regulator PPARG, that is probably related to the reported up regulated expression of the protein. Most importantly, these results also showed the upregulation of genes responsible for Clathrin (CLTC) and other adaptor-related proteins. An increase in CLTC expression in the transdifferentiated cells was confirmed by western blotting. Inactivation of CLTC by chlorpromazine (CHP), an inhibitor of CTLC mediated endocytosis (CME), and gene silencing by siRNAs, partially reversed the accumulation of neutral lipids observed in the transdifferentiated cells. These findings give a deeper insight into the phenotypic changes observed in HCCL to adipocyte-like transdifferentiation and point towards CME as a key pathway in distinct transdifferentiation programs.
Disclosures:
Simon C and Aguilar-Gallardo C are co-inventors of the International Patent Application No. PCT/EP2011/004941 entitled "Methods for tumor treatment and adipogenesis differentiation".
... Oil-Red Staining 0063 Lipid droplets were stained by Oil-red (Kutt and Tsaltas, 1959). A stock of Oil-red solution was prepared in 2-propanol (0.3%), Vortexed and filtered through a 0.8 um sieve before staining. ...
... 0071. To validate the accumulation of LDs after incubat ing cells with KOSR, we employed the Oil-RED dye that specifically binds to neutral lipids (Kutt and Tsaltas, 1959). ...
he present invention provides methods and pharmaceutical compositions for treating tumors, comprising administering to a subject with a tumor an amount effective of a formulation comprising one or more fatty acids selected from the group consisting of oleic acid [ (9Z) - Octadec - 9 - enoic acid], linoleic acid [cis, cis-9, 12 - octadecadienoic acid], palmitoleic acid [hexadec - 9 - enoic acid], and petroselenic acid [cis - 6 - octadecenoic], isomers thereof, and pharmaceutically acceptable salts thereof.
(FR)La présente invention concerne des méthodes et des compositions pharmaceutiques destinées au traitement de tumeurs, comprenant l'administration à un sujet souffrant d'une tumeur d'une quantité efficace d'une formulation contenant un ou plusieurs acides gras choisis dans le groupe constitué par l'acide oléique [acide (9Z)-octadéc-9-énoïque], l'acide linoléique [acide cis,cis-9,12-octadécadiénoïque], l'acide palmitoléique [acide hexadéc-9-énoïque] et l'acide pétrosélénique [acide cis-6-octadécénoïque], leurs isomères et leurs sels pharmaceutiquement acceptables.
... To corroborate a KOSR-dependent incubation accumulation of LDs, we employed the Oil-RED dye that specifically binds to neutral lipids [10]. HCCLs were cultured with HES+20%KOSR or HES+10%FBS for 48 h and were then stained by Oil-RED and hematoxylin ( Fig. 3a-o). ...
... Oil-RED staining Lipid droplets were stained by Oil-red [10]. A stock of Oilred solution was prepared in 2-propanol (0.3%), vortexed and filtered through a 0.8 μm sieve before staining. ...
Differentiation therapy pursues the discovery of novel molecules to transform cancer progression into less aggressive phenotypes by mechanisms involving enforced cell transdifferentiation. In this study, we examined the identification of transdifferentiating adipogenic programs in human cancer cell lines (HCCLs). Our findings showed that specific unsatturated fatty acids, such as palmitoleic, oleic and linoleic acids, trigger remarkable phenotypic modifications in a large number of human cancer cell lines (HCCLs), including hepatocarcinoma HUH-7, ovarian carcinoma SK-OV-3, breast adenocarcinoma MCF-7 and melanoma MALME-3M. In particular, we characterized a massive biogenesis of lipid droplets (LDs) and up-regulation of the adipogenic master regulator, PPARG, resulting in the transdifferentiation of HCCLs into adipocyte-like cells. These findings suggest the possibility of a novel strategy in cancer differentiation therapy via switching the identity of HCCLs to an adipogenic phenotype through unsaturated fatty acid-induced transdifferentiation.
... On coming in contact with the labile components of sweat residue, the stain partitions between the original solvent mixture, in which it is less soluble and the fats/lipids, in which it is more soluble. The molecules of ORO get preferentially dissolved in fat and lipid components and impart red color to these biomolecules (Kutt and Tsaltas 1959). The preferential solubility of ORO in fats and lipids is a physical phenomenon which is controlled by thermodynamic considerations. ...
Abstract Background Fingerprints are most frequently used to establish the identity of a person in medicolegal cases. Wide range of methods (optical, physical, and chemical) can be used to detect latent fingerprints on porous and non-porous items recovered from crime scenes. Oil Red O, also called solvent red 27, is a lipophilic dye, which means that it stains fat and lipid components in biological samples. It is also used to stain oil and waxes to a red hue. Oil Red O is used to detect latent fingerprints on dry and wet porous items like paper and cardboard. Result The reagent develops clear, stable, and red-colored fingerprints which may be discerned in natural light. Conclusion Although the physical developer can also lift latent impressions from wet porous surfaces, the method is a multistep one and requires immersion of delicate, paper-like articles in a sequence of working solutions. Compared to that, the operational steps of Oil Red O method are simple and cost effective and require less equipment to process items.
... Adipogenesis Adipogenic differentiation was induced by culturing FBs, sccFBs and PAs for 2 weeks in adipogenic medium ( table 1 ). Oil red O stain was used as an indicator of intracellular lipid accumulation [Kutt and Tsaltas, 1959]. Briefly, cells were rinsed in PBS, fixed for 60 min in 4% neutral buffered paraformaldehyde (NBP) and again rinsed in PBS. ...
The apparent need of an autologous cell source for tissue engineering applications has led researchers to explore the presence of cells with stem cell plasticity in several human tissues. Dermal fibroblasts (FBs) are easy to harvest, expand in vitro and store, rendering them plausible candidates for cell-based therapies. The aim of the present study was to observe the effects of adipogenic, chondrogenic and osteogenic induction media on the phenotype of human FBs. Human preadipocytes obtained from fat tissue have been proposed as an adult stem cell source with suitable characteristics, and were used as control cells in regard to their differentiation potential. Routine staining, immunohistochemical analysis and alkaline phosphatase assay were employed, in order to study the phenotypic shift. FBs were shown to possess multilineage potential, giving rise to fat-, cartilage- and bone-like cells. To exclude contaminant progenitor cells or cell fusion giving rise to tissue with adipocyte-, chondrocyte- and osteoblast-like cells, single-cell cloning was performed. Single-cell-cloned FBs (sccFBs) displayed a similar differentiation potential as primary-culture FBs. The presence of 'stem-cell-specific' surface antigens was analyzed using flow cytometry. The results reveal that sccFBs have several of the markers associated with cells exhibiting stem cell plasticity. The findings presented here are corroborated by the findings of other groups, and suggest the use of human dermal FBs in cell-based therapies for the reconstruction of fat, cartilage and bone.
... To investigate the effects of increasing Ca 2+ entry through SOCs on the differentiation of 3T3-L1 cells, confluent L1-STIM1 or L1-control pre-adipocytes were induced to differentiate by MDI and the extent of differentiation was quantified by staining lipid with Oil Red O (Kutt and Tsaltas, 1959). At the end of the 8-day differentiation period, both wild-type 3T3-L1 and L1-control cells had accumulated a comparable amount of lipid (Fig. 3A, top panels). ...
Ca(2+) plays a complex role in the differentiation of committed pre-adipocytes into mature, fat laden adipocytes. Stim1 is a single pass transmembrane protein that has an essential role in regulating the influx of Ca(2+) ions through specific plasma membrane store-operated Ca(2+) channels. Stim1 is a sensor of endoplasmic reticulum Ca(2+) store content and when these stores are depleted ER-localized Stim1 interacts with molecular components of store-operated Ca(2+) channels in the plasma membrane to activate these channels and induce Ca(2+) influx. To investigate the potential role of Stim1 in Ca(2+)-mediated adipogenesis, we investigated the expression of Stim1 during adipocyte differentiation and the effects of altering Stim1 expression on the differentiation process. Western blotting revealed that Stim1 was expressed at low levels in 3T3-L1 pre-adipocytes and was upregulated 4 days following induction of differentiation. However, overexpression of Stim1 potently inhibited their ability to differentiate and accumulate lipid, and reduced the expression of C/EBP alpha and adiponectin. Stim1-mediated differentiation was shown to be dependent on store-operated Ca(2+) entry, which was increased upon overexpression of Stim1. Overexpression of Stim1 did not disrupt cell proliferation, mitotic clonal expansion or subsequent growth arrest. siRNA-mediated knockdown of endogenous Stim1 had the opposite effect, with increased 3T3-L1 differentiation and increased expression of C/EBP alpha and adiponectin. We thus demonstrate for the first time the presence of store-operated Ca(2+) entry in 3T3-L1 adipocytes, and that Stim1-mediated Ca(2+) entry negatively regulates adipocyte differentiation. We suggest that increased expression of Stim1 during 3T3-L1 differentiation may act, through its ability to modify the level of Ca(2+) influx through store-operated channels, to balance the level of differentiation in these cells in vitro.
... The adipofibroblast cultures were stained for lipid content as previously described [Kutt and Tsaltas, 1959;Kinkel et al., 2004]. Briefly, the cultures were rinsed in PBS to remove medium components, and fixed in 10% formalin for 10 min. ...
Progeny adipofibroblast cells, derived from mature bovine adipocytes, were used to determine their ability to redifferentiate into lipid-assimilating adipocytes.
Traditional cell biology methods were used, including the expression of adipogenic markers such as peroxisome proliferator-activated receptor gamma (PPARgamma).
When exposed to medium supplemented with fetal bovine serum, but not horse serum, cells began to form structures reminiscent of foci. Horse serum-supplemented medium resulted in a slowed progression towards cell conversion to lipid-assimilating adipocytes. When analyzed, horse serum was found to contain more cortisol and insulin-like growth factor-1 as well as differing fatty acid ratios. Histological observations of the horse serum-treated cultures (alone), cultures treated with a traditional differentiation induction medium (dexamethasone, methylisobutylxanthine and insulin), treated with insulin with or without different lipid compounds, or treated with a PPARgamma agonist (rosiglitazone) resulted in the presence of intracellular vesicles, of which some contained lipid and some did not. Vesicles that did not stain for lipid did not possess glycogen or other types of storage moieties even though the cells expressed cellular markers thereby deeming them to be differentiated adipocytes (PPARgamma protein and mRNA were expressed by cells possessing vesicles as were hormone-sensitive lipase and lipoprotein lipase proteins). Non-lipid-filled intracellular vesicle walls possessed the structural protein perilipin.
These results are supportive of the progeny adipofibroblasts representing a unique adipogenic model that displays protracted adipogenesis.
The bluish-black spots of lipid-containing materials stained with a saturated solution of Sudan black B in 55% ethanol were found to fade and change color to brownish-pink shades in 5 min if exposed to ultraviolet light. Spots that were exposed to daylight for 6 hr on a sunny day lost 14% of their original color intensity but the decrease was less on cloudy days. Exposure to H2S initiated fading and color change in 2 hr. Exposure to HCl vapors restored the original color but not its intensity. Spots kept in darkness and wrapped airtight showed a decline of 2.5% in color intensity after 96 hr and no obvious color change. The speed and extent of change of color and fading of the various fractions of the dye separated by means of paper chromatography were different. Heat coagulated serum proteins were stained blue with commercial Sudan black B solution in 55% ethanol.
Saturated solutions of commercial Sudan II, III and IV in 60% ethanol were found to stain heat-coagulated fat-free purified human serum albumin and defatted whole serum. The protein staining components were isolated and identified by means of paper chromatography on mineral oil-impregnated filter paper with 95% ethanol as the developing agent. The brownish and yellowish fractions which migrated rapidly in this system stained proteins well, whereas the red, pink and orange fractions, which migrated slowly, stained lipids only. The solubility and staining power of the slowly migrating lipid coloring fractions remained satisfactory in absence of the rapidly moving protein staining fractions.
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