Effect of culture conditions on monoclonal antibody production from genetically modified tobacco suspension cultures

Oxygen supply and inoculum age were found to affect the production of the heavy chain monoclonal antibody (HC MAb) from genetically modified tobacco suspension cultures. The increase of oxygen supply increased both cell growth and HC MAb production. Furthermore, the increased aeration and mixing improved the production of HC MAb based on the unit amount of cells or total soluble proteins. This indicated that the increased aeration improved the production and secretion of HC MAb more than other cell components. HC MAb production and cell growth also improved when batch cultures were inoculated with actively dividing cells (5-day old) rather than the fullygrown cells (7- or 10-day old cells) that are commonly used for subcultures. The addition of glutamine to the medium also improved cell growth and HC MAb production.     

Hybridoma cell growth and monoclonal antibody secretion upon continuous cultivation in bioreactor with a serum- and protein-free nutritive medium

Summary 7F11C7 hybridoma cells, secreting a MAb useful for drug targeting, may be cultivated over more than 2 months in bioreactor upon continuous perfusion of serum-, protein-free nutritive medium. With optimal perfusion rate of 48%/day, in the absence of cell recycling, cell density reaches a mean of 2.85 10⁶ viable cells/ml (+ 7% of dead cells). MAb secretion reaches 78 g/ml x day, giving a production of 2.34 g/month x L, i.e. 13 times that obtained in T flasks.     

Saturated C21–C33Hydrocarbons Are Involved in the Self-Regulation of Pseudomonas fluorescensAdhesion to a Glass Surface

One of the two putative groups of antiadhesins was identified in Pseudomonas fluorescensby the method of gas chromatography–mass spectrometry. A mixture of high-molecular unbranched hydrocarbons (HC) with a chain length from 21 to 33 carbon atoms reduced cell adhesion to a glass surface. These HC accumulated in the culture liquid to a total concentration of 10–15 g/l; the concentrations of individual HC ranged from 0.1 to 3.0 g/l. After the addition of individual HC to the bacterial culture, the number of cells attached to the glass surface decreased. This decrease in cell adhesion was due to the enhanced aggregation of the bacterial cells, which promoted mechanical (hydrodynamic) cell detachment from the surface.     

Increase of hybridoma productivity using an original dialysis culture system

Hybridoma cell growth and monoclonal antibody production were investigated with a laboratory-made system in which cells were grown in dialysis tubing (MW cut-off 25 kD). The dialysis system contained 10 ml of cell suspension and was immersed in 200 ml of culture medium which when replaced or was at 4-day intervals. With this system, monoclonal antibody concentrations similar to those observed in ascites (concentrations in the order of one gramme per liter) were obtained. With no medium replacement, the antibody production was 3.3 g/l and the cell productivity 3.2×10^–8 g of IgM produced per cell in one minute. With medium replacement the antibody production was higher, 4.4 g/l but the cell productivity was lower, 1.49×10^–8 g per cell in one minute. Cells cultivated in non-optimized conditions were better producers than cells growing in a good environment.Abbreviations FCS fetal calf serum - Ig immunoglobulin - MAb monoclonal antibody - MW molecular weight - MWCO molecular weight cut off - RM replaced medium - NRM non replaced medium     

A new three-dimensional culture of human keratinocytes: optimization of differentiation

Many attempts have been made to obtain reconstructed human epidermis comprised of keratinocytes and extracellular-matrix constituents (essentially collagen) in the presence or absence of fibroblasts. A simple model of cultured human keratinocytes grown at the air-liquid interface of a noncoated artificial membrane, has been developed. This culture system offers many advantages: easy control of environmental factors and routine examination using optical or electronic microscopy, immunohistochemistry and indirect immunofluorescence techniques. This model enables the analysis of well-known differentiation markers and also intregrins, a family of cell-surface molecules involved in cell-cell and cell-extracellular matrix interactions, whose receptors are expressed on all basal keratinocytes.In our culture system, the expression of the different integrin subunits (2, 3, 5, 6, 1) was studied as a function of the differentiation state in two different media (K-SFM or DMEM/Ham's F12) supplemented with 5% fetal calf serum and adjusted to 1.5 mmol/L calcium. The most significant data are the preponderant expression of the 2 and 3 subunits in the basal and suprabasal layers, with membrane expression differing according to the culture medium; terminal differentiation was obtained in DMEM/Ham's F12. The use of membrane inserts represents a significant technological advance in culturing keratinocytes and is an easy-to-handle and valid model for determining the influence of physiological or pharmacological factors on cell proliferation or differentiation.Abbreviations DMEM Dulbecco's Modified Eagle's Medium - EGF epidermal growth factor - FCS fetal calf serum - K-SFM keratinocyte serum free medium - MAb monoclonal antibody - NHEK normal human epidermal keratinocytes - PBS Dulbecco's phosphate-buffered saline     

Enhancement of monoclonal antibody production by immobilized hybridoma cell culture with hyperosmolar medium

To determine the effect of hyperosmotic stress on the monoclonal antibody (MAb) production by calcium-alginate-immobilized S3H5/gamma2bA2 hybridoma cells, the osmolalities of medium in the MAb production stage were varied through the addition of NaCI. The specific MAb productivity (q(MAb)) of immobilized cells exposed to abrupt hyperosmotic stress (398 mOsm/kg) was increased by 55% when compared with that of immobilized cells in the control culture (286 mOsm/kg). Furthermore, this enhancement of q(MAb) was not transient. Abrupt increase in osmolality, however, inhibited cell growth, resulting in no increase in volumetric MAb productivity (r(MAb)). On the other hand, gradual increase in osmolality allowed further cell growth while maintaining the enhanced q(MAb) immobilized cells. The q(MAb) immobilized cells at 395 mOsm/kg was 0.661 +/- 0.019 mug/10(6) cells/h, which is almost identical to that of immobilized cells exposed to abrupt osmotic stress. Accordingly, the r(MAb) was increased by ca. 40% when compared with that in the control immobilized cell culture. This enhancement in i(MAb) of immobilized S3H5/gamma2bA2 hybridoma cells by applying gradual osmotic stress suggests the potential of using hyperosmolar medium in other perfusion culture systems for improved MAb production. (c) 1995 John Wiley & Sons, Inc.     

Product inhibition during the feeding phasein fed-batch ethanol fermentation of sugar-cane blackstrap molasses

Summary The following equations represent the influence of the ethanol concentration (E) on the specific growth rate of the yeast cells () and on the specific production rate of ethanol () during the reactor filling phase in fed-batch fermentation of sugar-cane blackstrap molasses: = ₀ - k · E and v = v ₀ · K/(K +E) Nomenclature E ethanol concentration in the aqueous phase of the fermenting medium (g.L^–1) - E_m value of E when = 0 or = 0 (g.L^–1) - F medium feeding rate (L.h^–1) - k empirical constant (L.g^–1.h^–1) - K empirical constant (g.L^–1) - M_as mass of TRS added to the, reactor (g) - M_cs mass of consumed TRS (g) - M_e mass of ethanol in the aqueous phase of the fermenting medium (g) - M_s mass of TRS in the aqueous phase of the fermenting medium (g) - M_x mass of yeast cells (dry matter) in the fermenting medium (g) - r correlation coefficient - S TRS concentration in the aqueous phase of the fermenting medium (g.L^–1) - S_m TRS concentration of the feeding medium (g.L^–1) - t time (h) - T temperature (° C) - TRS total reducing sugars calculated as glucose - V volume of the fermenting medium (L) - V₀ volume of the inoculum (L) - X yeast cells concentration (dry matter) in the fermenting medium (g.L^–1) - filling-up time (h) - specific growth rate of the yeast cells (h^–1) - ₀ value of when E=0 - specific production rate of ethanol (h^–1) - ₀ value of when E=0 - density of the yeast cells (g.L^–1) - dry matter content of the yeast cells     

An immobilized hybridoma culture perfusion system for production of monoclonal antibodies

A fixed bed perfusion system for hybridoma cell immobilization is presented. The system consists of a culturing vessel (300 ml total volume) in which polyurethane (PU) sponges in the form of small cubes of about 5 mm sides are packed. Cells are immobilized by physical entrapment in the foam matrix. By entrapment of the cells in the pores of the matrix high cell concentration can be maintained in a mechanically protected environment. Medium is continuously circulated by an airlift pump mounted in the cell-free chamber (700 ml total volume).Medium flow rate, feeding rate, dissolved oxygen, pH, nutrient uptake and waste product formation can be easily monitored and controlled. Steady state conditions are established with medium dilution rates of 1.0–1.5 reactor volume per day. The steady state is characterized by a constant cell density, constant culture volume and constant glucose and lactate levels. Cell-free supernatant is collected continuously in a cold room adjacent to the 37°C culture room. Monoclonal antibodies (MAb) are produced at a concentration of 150–200 g/ml for several weeks. An important feature of the system is the capacity to maintain a population of cells after the growth phase in a non-proliferating state for extended time periods expressing high titers of MAb.Abbreviations DO Dissolved Oxygen - FBS Fetal Bovine Serum - FBR Fixed Bed Reactor - MAb Monoclonal Antibody - PU polyurethane     

Killer toxin secretion through the cell wall of the yeastPichia anomala

A secreted killer toxin was detected through the cell wall ofPichia anomala cells by ultrastructural immunodetection with a specific monoclonal antibody (MAb KT4). MAb KT4 was successively detected by colloidal gold labeled streptavidin and biotinylated anti-mouse F(ab')₂ antibodies. The antigenic determinants of the toxin were localized throughout the cytoplasm and the cell wall of killer yeast cells. The Lowicryl K4M-immunogold method gave very satisfactory results and showed that the killer toxin was somewhat concentrated in the yeast cell wall layers before being exported into the medium. In agreement with previous reports, the binding of MAb KT4 suggested that theP. anomala killer toxin secretion did not result from a homogeneous diffusion across the yeast cell wall.Abbreviations G15 gold particles of 15 nm - IEM immunoelectron microscopy - IFA immunofluorescence assay - MAb monoclonal antibody - PBS phosphate buffered saline - SAM/F(ab)₂ sheep antibodies anti-mouse F(ab)₂ - SBB Sabouraud buffered broth     

Observations consistent with autocrine stimulation of hybridoma cell growth and implications for large-scale antibody production

Existence of autocrine growth factors (aGFs) may influence the serum requirement for growth of hybridoma cells and thus significantly influence process economics. For the murine hybridoma cell line S3H5/2bA2, critical inoculum density (cID) and serum requirement for growth were inversely related for cultivation in both T flasks and spinner flasks. In spinner flasks, an inoculum density of 10⁶ cells/ml was necessary for the cells to grow in RPMI 1640 medium without serum supplement, and an inoculum density of 10³ cell/ml was necessary in RPMI 1640 medium with 10% serum. In T flasks, where the local cell density is higher than in spinner flasks, an inoculum density of 10⁶ cells/ml was necessary for the cells to grow in RPMI 1640 medium without serum supplement, and an inoculum density of 1 cell/ml was also necessary in RPMI 1640 medium with 10% serum. Further, immobilized cells at high local cell density could grow under conditions where cells in T flasks at corresponding overall cell density could not grow. The cells at high inoculum density were less sensitive to shear induced by mechanical agitation than the cells at low inoculum density. Taken together these observations support the existence of secreted aGF(s) by the hybridoma cell line used. Since the specific MAb production rate was independent of cultivation method and inoculum density, the existence of autocrine growth factors would suggest that the use of immobilized cells should improve the economics of MAb production.     

An Antibody against a Novel and Conserved Epitope in the Hemagglutinin 1 Subunit Neutralizes Numerous H5N1 Influenza Viruses

The spread of the recently emerged, highly pathogenic H5N1 avian influenza virus has raised concern. Preclinical studies suggest that passive immunotherapy could be a new form of treatment for H5N1 virus infection. Here, a neutralizing monoclonal antibody (MAb) against the hemagglutinin (HA) of the influenza A/chicken/Hatay/2004 H5N1 virus, MAb 9F4, was generated and characterized. MAb 9F4 binds both the denatured and native forms of HA. It was shown to recognize the HA proteins of three heterologous strains of H5N1 viruses belonging to clades 1, 2.1, and 2.2, respectively. By use of lentiviral pseudotyped particles carrying HA on the surface, MAb 9F4 was shown to effectively neutralize the homologous strain, Hatay04, and another clade 1 strain, VN04, at a neutralization titer of 8 ng/ml. Furthermore, MAb 9F4 also neutralized two clade 2 viruses at a neutralizing titer of 40 ng/ml. The broad cross-neutralizing activity of MAb 9F4 was confirmed by its ability to neutralize live H5N1 viruses of clade 2.2.2. Epitope-mapping analysis revealed that MAb 9F4 binds a previously uncharacterized epitope below the globular head of the HA1 subunit. Consistently, this epitope is well conserved among the different clades of H5N1 viruses. MAb 9F4 does not block the interaction between HA and its receptor but prevents the pH-mediated conformational change of HA. MAb 9F4 was also found to be protective, both prophylactically and therapeutically, against a lethal viral challenge of mice. Taken together, our results showed that MAb 9F4 is a neutralizing MAb that binds a novel and well-conserved epitope in the HA1 subunit of H5N1 viruses.The highly pathogenic avian influenza A subtype H5N1 virus was first isolated from geese in Guangdong province, China, in 1996 (44). Since 2003, the H5N1 strains have caused major morbidity and mortality in poultry populations across Asia, Europe, and Africa (3, 25). In 1997, the virus was transmitted from chickens to humans in Hong Kong, causing 18 reported cases of illness, including 6 deaths (6, 7, 37). As of September 2009, there were 442 confirmed human infections in 15 countries, with an alarming fatality rate of 59% (42). Although occurrences of human H5N1 infection are sporadic and rare, its rapid dissemination, the ongoing evolution of the avian H5N1 virus, and the absence of anti-H5N1 herd immunity in humans raise concerns regarding a possible H5N1 influenza pandemic (2, 4, 13). Since human infections are associated with severe disease and high mortality, the consequences of a pandemic could be catastrophic.Current strategies against influenza include vaccination and antiviral drug treatment (1). Due to the existence of multiple antigenic clades and subclades of the H5N1 virus, the difficulty of predicting the major strain that may cause the next pandemic is the main obstacle to current vaccine development. Moreover, resistance to M2 ion channel inhibitors (rimantidine and amantidine) has been reported in H5N1 isolates (1, 5), and the neuraminidase inhibitors (oseltamivir and zanamivir) require higher doses and prolonged treatment (45), and resistance has been reported in children (21). Passive immunotherapy is now increasingly used to treat numerous human infectious diseases (28, 33). Convalescent-phase blood and serum products were used to improve clinical outcomes for severely ill influenza patients during the 1918 influenza pandemic (27). Promising results with mouse models using a neutralizing monoclonal antibody (MAb) for H5N1 influenza treatment (17, 26) and a report of the recovery of an H5N1 virus-infected patient after treatment with convalescent-phase plasma (47) indicate that MAbs could be a potential treatment against H5N1 viruses.The hemagglutinin (HA) protein is one of the two major surface glycoproteins on the envelope of influenza A virus, with 16 distinct types identified in the avian species. The HA protein is responsible for receptor binding to host cells and for viral entry and is therefore the primary target of neutralizing antibodies (Abs) (35). It is a homotrimer, with each subunit made up of two disulfide-linked polypeptides, HA1 and HA2. Structurally, each subunit consists of a membrane-proximal helix-rich stem structure and a membrane-distal receptor binding globular domain (35).In this study, we describe a MAb, named MAb 9F4, raised against the recombinant baculovirus-expressed HA protein of A/chicken/Hatay/2004 H5N1 virus. Its neutralizing property was investigated, and epitope mapping was performed. The MAb 9F4 binding site was found to lie outside previously characterized antigenic sites in the HA protein. This epitope is well conserved among the different clades of H5N1 viruses, consistent with the cross-neutralizing activity of MAb 9F4. The mode of inhibition was also investigated, and MAb 9F4 was found to mediate postattachment neutralization in a dose-dependent manner. Finally, the protective ability of MAb 9F4 was also evaluated in a mouse model, and it was shown to protect against lethal H5N1 challenge both prophylactically and therapeutically. Taken together, the data could provide new information for the design of an H5N1 vaccine, and MAb 9F4 may be a possible candidate for use in passive immunotherapy.     

Comparison of changes in toxicity during growth of Anabaena circinalis (cyanobacteria) determined by mouse neuroblastoma bioassay and HPLC

Changes in toxicity were studied during batch culture of a strain ofAnabaena circinalis (AWQC ANA-311F) producing paralyticshellfish toxins (PSTs). PSTs were extracted from cells and culture mediumduring the culture period of 29 days. Samples were analysed by HPLC withpost-column oxidation coupled to fluorescence detection, and tested with themouse neuroblastoma bioassay (MNB). Seven PSTs were detected, but only fivewerequantified. Based on chemical analysis of PSTs by HPLC, atheoretical saxitoxin concentration related to mouse toxicity wascalculated. Neuroblastoma cell survival was measured during the MNB andcorrelated to total toxicity of the A. circinalis samples.Comparison was made between the changes in total toxicity shown by MNB and thetheoretical saxitoxin concentration shown by HPLC. There was asignificant positive correlation between the two in the culture medium, but notthe cells.     

Mitochondrial activity of 2,6-diaminopurine in Saccharomyces cerevisiae.

Summary 2,6-diaminpurine (DAP) selectively inhibited mitochondrial protein synthesis in yeast cells with concomitant failure of cells to grow in non-fermentable (yeast extract, glycerol) medium. The selectivity was pronounced in all strains tested (15) nearly all of which were able to grow in yeast extract, glucose medium containing 5 mg/ml DAP (maximum solubility) whereas growth was arrested in all strains at 250–500 g/ml DAP in the glycerol medium. The inhibition was reversed by further addition of adenine to the culture medium. RNA synthesis in rat liver mitochondria was depressed by DAP suggesting that the analogue affected RNA polymerase activity.There was no evidence of nuclear mutagenicity by DAP but resistance to the antibiotics chloramphenicol and oligomycin was induced by the drug. Genetic evidence, although limited, indicated that the resistance mutations were cytoplasmic. The mitochondrial petite mutation was also induced by DAP but only at comparatively high concentrations. The mutagenic effects were seen only in the glycerol medium.     

Human Aurora-B binds to a proteasome α-subunit HC8 and undergoes degradation in a proteasome-dependent manner

Human Aurora/Ipl1-related kinase 2 (Aurora-B) is a key regulator of mitosis. Here human proteasome -subunit C8 (HC8) was identified to interact with the Aurora-B by yeast two-hybrid screen. This finding was confirmed by GST pull-down assays and immunoprecipitation experiments. The Aurora-B protein level increased in HeLa cells cultured with proteasome inhibitor ALLN. Our data suggest that Aurora-B might undergo degradation by binding to HC8 in a proteasome-dependent manner during mitosis.     

Hormonal regulation of berberine production in cell suspension cultures of Thalictrum minus

Effects of auxin and cytokinin on cell growth and alkaloid production in cell suspension cultures of Thalictrum minus were examined in an attempt to increase the productivity of a medicinal compound, berberine. In Linsmaier and Skoog medium containing auxin such as 2,4-D (1 M), the cultured cells grew rapidly, producing little berberine. On the other hand, the berberine-producing activity was remarkably enhanced by simultaneous administration of auxin and cytokinin, although cell growth was inferior. In particular, for the combination of NAA (60 M) and 6-benzylaminopurine (10 M), the yield of berberine was as high as 20 mg/30 ml medium after 2 weeks of culture. Furthermore, most of the berberine produced by the cells was released into the liquid medium, in which an excess of berberine crystallized. The results of the present experiments are suggestive of an advantage in adopting a two-stage culture method for the production of berberine in fermentor systems.Abbreviations LS Linsmaier and Skoog - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - BAP 6-benzylaminopurine     

Phorbol ester stimulation of phosphatidylcholine synthesis in four cultured neural cell lines: Correlations with expression of protein kinase C isoforms

Phosphatidylcholine (PtdCho) can provide lipid second messengers involved in signal transduction pathways. As a measure of phospholipid turnover in response to extracellular stimulation, we investigated differential enhancement of [³H]choline incorporation into PtdCho by phorbol esters. In C6 rat glioma and SK-N-SH human neuroblastoma cells, [³H]PtdCho synthesis was 2–4 fold stimulated by -12-O-tetradecanoylphorbol-13-acetate (-TPA) when [³H]choline was incubated simultaneously with, or 15 min prior to, -TPA treatment. By contrast, in N1E-115 mouse and SK-N-MC human neuroblastoma cells, phorbol esters had no appreciable effect on [³H]choline incorporation; however, in all cells, 200 M oleic acid enhanced PtdCho synthesis, indicating a stimulable process. Alterations by thymeleatoxin (TMT), an activator of conventional PKC isoforms (, and ), were similar to -TPA. We investigated whether expression of specific PKC isoforms might correlate with these effects of phorbol esters on PtdCho synthesis. All cell lines bound phorbol esters, had PKC activity that was translocated by phorbol esters and differentially expressed isoforms of PKC. Northern and western blot analyses, using specific cDNA and antibodies for PKC-,-,-,-,-, and-, revealed that expression of -isoform predominated in C6 and SK-N-SH cells. In contrast, TPA-responsive -isoform predominated in SK-N-MC cells. -PKC was not detected in any cells and only in C6 cells was PKC- present and translocated by -TPA treatment. PKC- was not detected in SK-N-MC cell lines but translocated with TPA treatment in the other three cell lines. PKC- was present in all cells but was unaltered by TPA treatment. Accordingly, stimulation of PtdCho turnover by phorbol esters correlated only with expression of PKC-; presence of PKC- alone was insufficient for a TPA response.Abbreviations DAG diacylglycerol - DMEM Dulbecco's modified Eagle's medium - dPPA 12-deoxyphorbol-13-phenylacetate-20-acetate - PKC protein kinase C - cPKC conventional PKC - PtdCho phosphatidylcholine - TPA 12-O-tetradecanoylphorbol-13-acetate - TMT thymeleatoxin     

Efficient production of recombinant protein in Spodoptera frugiperda/AcNPV system utilizing silkworm hemolymph

Silkworm hemolymph when added at 5% (v/v) to medium increased the production of recombinant -galactosidase in Spodoptera frugipera/ Autographa californica nuclear polyhedrosis virus (AcNPV) system by 4.5-fold. Silkworm hemolymph also increased the host cell longevity by two-fold. Viability of the host cell was maintained at a constant level for 6 days after baculovirus infection in the medium containing 5% silkworm hemolymph, while host cells began to die 3 days after infection in the medium without hemolymph.     

Long term and large-scale cultivation of human hepatoma Hep G2 cells in hollow fiber bioreactor

Long-term and large scale cultivation of an anchorage-dependent cell line using an industrial scale hollow fiber perfusion bioreactor is described. Hep G2 cells (a human hepatoma cell line) were cultivated in an Acysyst-P^® (Endotronic) with a total fiber surface area of 7.2 m² (6×1.2 m²) to produce Hep G2 crude conditioned medium (CCM). Pretreatment of the cellulose acetate hollow fibers with collagen enhances the attachment of the anchorage-dependent cells. We have succeeded in growing the Hep G2 cells in an antibiotics-and serum-free IMDM medium, supplemented with 50g/ml of Hep G2 CCM protein at inoculation. The Hep G2 cells replicate and secrete CCM protein in quantities comparable to those produced in DMEM containing 10% fetal calf serum (FCS). The highest CCM protein productivity during the 80-day cultivation was 1.1 g/day with a total of 30 g of protein accumulated. Hep G2 CCM (20–40 g protein/ml) was comparable to or even better than 10% FCS in supporting the growth of Molt-4 (a human T leukemia cell line) and FO (a mouse myeloma cell line) cells in vitro. The availability of this large amount of Hep G2 CCM will aid the further purification and characterization of growth factor(s) which could be used as serum substituents.     

Decrease of P-Glycoprotein Activity in K562/ADR Cells by MβCD and Filipin and Lack of Effect Induced by Cholesterol Oxidase Indicate That This Transporter Is Not Located in Rafts

The effect of low-density membrane domains on function of the plasma membrane transporter P-glycoprotéine (P-gp), involved in multidrug resistance (MDR) phenotype, has been investigated in K562/ADR cells. To this end we reversibly altered the cholesterol content of K562/ADR cells by using methyl--cyclodextrin as a cholesterol chelator and conversely we repleted them through incubation with cholesterol in culture medium. We also used the cholesterol-binding fluorochrome filipin and cholesterol oxidase. Our data show that either cholesterol depletion or complex formation with filipin resulted in a strong decrease of P-gp activity. However, when cells were incubated with cholesterol oxidase that are known to disrupt rafts, no modification of the P-gp activity was observed. In addition, using a free-detergent methodology to separate by ultracentrifugation, light, heavy, and extra heavy fractions we show that no P-gp is found in the light fraction where rafts are usually detected. Altogether, our data strongly suggest that, in this cell line, P-gp is not localized in rafts.