How does dmem taste

We used Giga-seal whole cell recording to examine the membrane properties of cells in cultured taste buds to compare them with the properties of cells in acutely isolated taste buds. Seals of several gigaohms were readily obtained from cultured cells from all times tested. Interestingly, whole cell recordings were obtained much more readily from the cultured cells than from freshly isolated cells, where balloons of membrane are often sucked into the pipette during attempts at seal formation.

Membrane currents were elicited by stepwise depolarizing pulses from a holding potential of —80 mV. After 7 days the cultures were quite variable, with some cultures still showing taste cells with voltage-gated currents see for example Figure 4 and other cultures showing few if any taste cells with voltage-gated currents. Resting potentials were consistent in the cultures Table 1 , but the values were somewhat lower than what is typically found in freshly isolated taste buds Behe et al.

We observed that with time in culture an increasing number of taste cells would fail to seal well and thus would exhibit large leak conductances. In some cells membrane capacitance increased as well, suggesting that cells may be electrically coupled in older cultures. We obtained recordings from cultured circumvallate, foliate and fungiform taste cells Figure 4 , however, we analyzed in detail only currents from circumvallate taste buds Table 1.

It was difficult to obtain good seals and non-leaky whole cell recordings. However, these proteins are detectable only in differentiated taste cells and disappear within 7—14 days following denervation of taste buds in the intact animal McLaughlin et al.

Isolated taste buds in culture are completely separated from their nerve supply. Thus, an interesting question is whether cultured taste buds retain cellular and surface proteins that are affected by denervation in vivo. Cells in cultured taste buds exhibited immunoreactivity for each of the markers tested and the approximate number of labeled cells per bud was similar to that seen following fixation of an intact animal Smith et al. Gustducin immunoreactivity was observed in taste buds cultured for up to 14 days at room temperature, with no apparent differences in taste buds isolated from the different papillae data not shown.

Immunoreactivities to PGP 9. Immunocytochemical analysis became problematical after longer periods because the taste buds detached from the substrate during processing. To test whether cultured taste buds contained proliferative cells BrdU was added to the cultures immediately following isolation and 24 h after isolation. The BrdU remained in contact with the cultures for up to 72 h to increase the probability of labeling slowly dividing cells. When BrdU was added to the culture medium at the time of plating and buds were cultured at room temperature for 2 days in the presence of BrdU some taste buds had as many as 5—8 labeled nuclei following immunocytochemical processing Figure 7 A,B.

Addition of BrdU to taste buds that already had been in culture for 48 h resulted in essentially no BrdU label. Thus, cell division continues in culture immediately following isolation, but then either stops or is reduced to a rate undetectable by the short-term techniques employed in our studies. Following fixation and immunocytochemical processing taste buds were analyzed for BrdU incorporation. Only a small number of taste buds exhibited BrdU labeling and only 1—2 cells were labeled per bud Figure 7 C.

In some cases it was not clear whether the labeled cells were part of the taste bud or associated non-taste cells. These cultured taste buds retain electrophysiological and immunocytochemical properties and individual cells within the buds maintain elongated morphologies. In addition, some cells in isolated taste buds are able to divide when initially placed in culture.

In our studies taste buds were separated from their normal microenvironment. Major components that were lost during this process included surrounding epithelial cells, associated nerve fibers and saliva. In place of these elements a defined medium was used. We did not expect this medium to replicate in vivo conditions. The goals of these experiments were to establish a defined culture system for maintaining differentiated taste buds for short periods of time, to describe the characteristics and changes exhibited by taste buds in culture and to provide a general time course for these changes.

Ultimately, the global purposes of these studies were to create a short-term culture system for expressing foreign proteins and to provide a starting point for investigating specific components e. Under both temperature conditions the taste buds demonstrated the same sequence of events, although the timing of these events differed dramatically. Room temperature culture likely slows down most cellular processes, including degeneration.

This idea is consistent with studies in catfish indicating that decreasing temperatures increase taste bud survival times following denervation Torrey, and that the average lifespan of a taste cell is temperature dependent Raderman-Little, When cultured at room temperature isolated taste buds maintained most of their phenotypic properties for 10—14 days. Some taste buds were lost prior to day 10, but this was likely due to detachment from the substrate or damage during isolation and plating.

With the exception of these damaged taste buds, the most obvious change prior to day 10 was rounding up of the taste buds, which occurred soon after plating.

Despite this change in overall shape of the cultured end organ, individual taste cells retain an elongated morphology. The two temperatures produced no obvious differences in timing with respect to this overall change in taste bud shape.

The next observed change was degeneration and loss of peripheral cells from taste bud aggregates. Sloughing of dead cells from the compact taste bud in culture may mimic the extrusion of dead or dying taste cells from the bud in vivo Guth, These central cells excluded Trypan blue, exhibited immunoreactivity for specific proteins and had voltage-gated currents.

Elongated taste cells were apparent in the taste bud clusters throughout these time periods. In contrast to the apparent limited effect of culture on differentiated taste cells, proliferative cells associated with taste buds were quickly and dramatically affected by isolation and culture. The labeling of cells in taste buds at the initial time points indicates that proliferative cells were included in the cultures and that isolation and culture resulted in an inability of these cells to divide at later times.

Specific environmental features appear to be important for taste bud survival in the intact animal and loss of these associations in culture likely contributed to the decline of taste bud health in cultures over time. Denervation studies in vivo indicate that loss of innervation results in dramatic decreases in taste cell number within 4—5 days following nerve injury and complete loss of normal taste cells 7 days after surgery Guth, ; Farbman, In addition, Smith et al.

Thus, taste bud loss following denervation is a gradual process and resembles changes seen in cultured taste buds. Although most recognizable taste buds are gone following 14 days in room temperature cultures, a few taste buds remain. The degree and timing of taste cell loss in vivo following denevation is dependent on several factors, including the papilla examined and the length of nerve left attached to the taste bud.

In the intact animal fungiform buds are affected less by denervation than other lingual taste buds Whitehead et al. We see no obvious differences in survival in vitro of the taste buds from different papillae. However, fungiform taste buds are difficult to isolate, resulting in fewer healthy taste buds at the start of culture relative to circumvallate and foliate cultures. This makes direct comparisons of culture viability between the different types of taste buds difficult.

Denervation studies in intact animals indicate that nerve fragments associated with taste buds influence taste bud survival Torrey, ; State and Dessouky, During denervation experiments the nerve is typically sectioned or crushed proximal to the lingual epithelium. This results in the retention of nerve fragments associated with taste buds distal to the nerve injury.

State and Dessouky compared the length of the remaining nerve fragment with taste bud survival times and found that longer nerve fragments resulted in slower degeneration of taste buds from the injury State and Dessouky, In contrast to denervation studies in the intact animal, in culture there is a complete and uniform removal of innervating fibers except for the extreme distal processes ramifying between the taste cells.

However, this is a qualitative observation complicated by extensive variability in the cultures. If this observation is accurate, the quicker demise may be due to complete loss of innervation or to the combined loss of several elements in culture.

Cultured taste buds not only lack innervation, but are missing other factors and tissue relationships as well. For example, cultured taste buds must survive in the absence of saliva. Removal of salivary glands adversely affects taste buds in the intact animal and these changes have been attributed largely to the loss of EGF, which occurs in high concentrations in the saliva Cano and Rodriguez-Echandia, ; Nanda and Catalanotto, , Morris-Wiman et al.

Epithelium, connective tissue and other lingual cells may also contribute to the health of a normally functioning taste bud. For example, Suzuki et al. Cells from surrounding tissue, most likely fibroblasts, reportedly phagocytose dying taste buds following denervation Suzuki et al. Despite our attempts to produce pure taste bud cultures, some non-taste cells are present.

No attempt was made to specifically identify these cells, but they might have been fibroblasts. Their proliferation at this temperature may be a factor in the shorter survival of the cultured taste buds. In summary, we have developed a culture system for maintaining differentiated adult taste buds in vitro. Removal of Noggin from culture medium leads to loss of Lgr5 expression and proliferation arrest in cultured intestinal organoids More taste cells e.

A similar pattern was observed for Lgr5 and the proliferating cell marker Ki67 Fig. S6B, C. Our immunostaining results showed that a great number of cells in organoids grown under both conditions were immunoreactive for taste cell markers. These data suggest that Noggin plays an essential role in taste cell differentiation and stem cell proliferation in the cultured taste organoids.

Noggin promotes the proliferation and differentiation of taste organoids. The number of organoids containing Gad1-GFP cells increased at both day 10 and day 14, although this was not statistically significant Fig. These data suggest that blocking Notch signaling accelerates the differentiation of mature taste cells and alters cell fate determination as well.

Dibenzazepine DBZ accelerates and promotes differentiation of taste cells in cultured organoids. A Schematic illustration of DBZ treatment on cultured organoids at indicated days. D Quantitative real-time PCR analysis of the relative expression level of genes specific for proliferation, stem cells, and taste cells in control and DBZ-treated organoids.

To determine if Hh signaling is required for the growth and differentiation of taste organoids, we used a specific Gli transcriptional factor inhibitor, GANT61, to block Hh signaling during different stages of organoid growth To distinguish between these two possibilities, we removed GANT61 at day 5 to determine if cells would proliferate and generate organoids after the removal of GANT We observed that single cells started to grow into organoids after removal of GANT61, and these organoids included mature taste cells at day 14 Fig.

Furthermore, long-term incubation with GANT61 from day 0 to day 10 inhibited the growth of organoids, which had much smaller size and fewer taste cells Fig. GANT61 arrests the growth of taste organoids. B Immunostaining of day organoids with anti-Car4 antibody without top row or with lower rows GANT61 treatment at different stages. C Quantitative real-time PCR analysis of the relative expression level of genes specific for proliferation, stem cells, and taste cells in control and GANTtreated organoids.

Quantitative analysis performed at day 14 showed that both proliferating cell marker Ki67 and mature taste cell markers Gustducin and Snap25 were significantly downregulated by GANT61 from day 0 to day 10 Fig.

However, 10 days of incubation with GANT61 in a later stage from day 4 to day 14 had much less of an effect Fig. These organoids had fewer taste cells and smaller size than controls but more taste cells and larger size than those incubated with GANT61 from day 0 to day 10, suggesting that blocking Shh pathway by GANT61 arrested the proliferation of stem cells in cultured taste organoids, especially in the early stage of culturing.

Interestingly, the level of Lgr5 expression significantly increased in organoids treated with GANT61 from day 4 to day 14, suggesting an interplay between Wnt signaling and Hh signaling.

We analyzed the transcriptomes of taste organoids at different stages of development and found that in organoids at earlier stages multiple cell-cycle-related genes are activated, including those for M phase, cell cycle, mitosis, and cell division, suggesting that active proliferation occurs in the earlier organoids. At later stages e. Moreover, we found that modulation of Notch signaling accelerates the maturation of taste receptor cells in taste organoids.

We combined RNA-Seq with a novel culture system to uncover genes and pathways involved in the ontogeny and cell-fate determination of taste cells. Different waves of transcriptional activity were evident at different stages of development of taste organoids, suggesting specific and fine regulation of genetic networks in the patterning and generation of taste receptor cells.

For instance, transcripts of transcription factors that are known to specify taste cell determination e. Detailed analyses of these upregulated genes e.

We showed that several signaling pathways are involved in the growth and differentiation of taste organoids. Hh signaling is implicated in taste tissue homeostasis 4 , 15 , Not surprisingly, we found that the blockade of Gli transcription led to the arrest of growth of taste organoids. In developing taste tissue, Shh negatively regulates Wnt activity It is noteworthy that upregulation of Lgr5 transcripts occurred by blockage of Shh signaling during day 4—14 of culture Fig.

Inhibition of Wnt signaling ablated long-term organoid cultures Here, we found that organoid growth and taste cell generation were Wnt activity dependent. Noggin activity can affect the growth of organoids as well as taste cell generation. Because of their increased expression as organoids mature, a subset of genes related to Notch signaling e. Notch signaling is implicated in multiple developmental processes 32 , 41 , 43 and in taste tissue differentiation and homeostasis 32 , However, the role of Notch signaling in mammalian taste development and regeneration is largely unexplored.

This indicates that Notch signaling may be involved in taste cell fate determination. Most likely, the increase in the number of type II and type III taste cells is due to fate alteration of type I cells in cultured taste organoids in the presence of DBZ. Future work using knockout models and organoid cultures may provide a mechanistic understanding of the Notch signaling pathway in taste tissue homeostasis. Multiple chemotherapy agents are known to affect taste function, presumably by affecting taste cell regeneration.

Given the effects of GANT61 on taste cell proliferation and recycling, this culture system may be used as a prescreening tool to evaluate the potential side effects of cancer therapy drugs. Previous attempts to identify receptors, channels, and key signaling elements of taste transduction used cDNA libraries to identify genes enriched in taste tissue 45 , Although these methods helped identify the receptors for sweet and umami 47 , the detectors responsible for transducing high salt, sour, and some other unconventional tastes remain elusive.

RNA-Seq analysis of taste organoids provides a temporal dimension to the identification of candidate genes relevant for taste transduction. In our dataset, most known taste receptors and signaling elements appear only in late-stage organoids cluster 1. Thus, the transducers for high-salt, sour, and other tastes may also be expressed only in late-stage organoids. Analysis of the function of late-expressed channels or receptors may help reveal the identity of additional taste receptors. The growth and differentiation of cultured organoids appear to largely mimic the in vivo regeneration of native taste bud cells.

Nevertheless, our cultured organoids differ from native tissue. For instance, we observed the expression of only a subset of Tas2r bitter receptor genes in our dataset and failed to detect many other Tas2rs, possibly because a their expression level is below our detection limit due to the depth of sequencing—indeed, it appears that those receptors are only weakly expressed in native taste tissue 48 ; b organoids require more time for maturation in our system; c our system cannot entirely capture everything happening in native taste buds; or d technical difficulties in amplifying all relevant genes from our organoids due to a limited amount of starting material and potential biases during the amplification of cDNA for sequencing.

Despite a great number of mature taste cells in our cultured organoids, we did not observe well-organized taste bud-like structures in vitro , suggesting some limitation of our cultured system. Further optimization is required for growing a fully functional bud in vitro. Trpm5-GFP mice were generated in the Margolskee lab. All experiments were performed under National Institutes of Health guidelines for the care and use of animals in research and approved by the Institutional Animal Care and Use Committee of the Monell Chemical Senses Center.

Tongue epithelium was peeled gently off from the underlying connective tissue, and the regions surrounding the circumvallate and foliate papillae were dissected out and collected. Tissues were then minced using scissors and digested by trypsin 0.

Cells were dissociated from the circumvallate papilla tissue from at least three mice. The expression of Noggin was confirmed by immunostaining. The medium was first changed after 5 days of culturing and then every 2—3 days based on the density of growing organoids. Wnt activity was determined by TOP-flash assay, as described previously These reporter constructs were kindly shared by Dr. A pRL Renilla luciferase control reporter vector Promega served as a control for transfection efficiency.

Cells were lysed, and cell lysates were assayed for firefly luciferase and Renilla luciferase activity on a luminometer using the Dual-Luciferase reporter assay system Promega, ref E Libraries were sequenced on an Illumina HiSeq to generate bp reads. Statistical tests based on a negative binominal generalized linear model GLM similar to that of edgeR were conducted in CLC genomics workbench 9.

We used likelihood ratio tests ANOVA-like tests to estimate differential gene expression across days while controlling for difference between replications. For K-means clustering, the trimmed datasets see main text were log transformed, and then the reads for each gene across all different stages were normalized to the average of reads of all different stages for each gene.

For hierarchical analysis, we used the hclust and heatmap. Immunostaining of organoids was performed in 1. Organoids in mounting medium were then transferred onto slides for imaging Prolong Gold Antifade Mountant, Thermo Scientific. Secondary antibodies were donkey anti-rabbit: Alexa Fluor Abcam no. Fluorescence images were acquired by a Nikon Eclipse E microscope. In vivo , the normal concentration of mouse blood serum insulin is around Although in many cell culture studies, a high concentration of insulin was applied to visualize its effect, for example, to detect its proliferative effect e.

The taste bud organoid system mimics many aspects of taste organ growth ex vivo but still could not completely reproduce taste tissue and the surrounding environment. To elucidate the relationships among various growth factors, including insulin might be the next step to advance this system.

However, no significant change was observed in GLUT4 expression Fig 3 , perhaps because GLUT4 expression is not restricted to taste cells but also is frequently expressed in NT or mesenchymal cells [ 16 ].

In our experimental setting organoid colonies were derived from mouse tissue surrounding CV, containing taste progenitor cells and NT progenitor cells, so some colonies might have contained NT tissue, which would make it difficult to see the change in GLUT4 expression specifically in taste cells.

Moreover, mTOR inhibition is reported to ameliorate radiation-induced salivary gland damage [ 32 ]. A rapamycin analogue induced autophagy and then suppressed an exacerbated compensatory proliferation, which allowed for improvement and reestablishment of salivary gland function [ 32 ]. Autophagy is a homeostatic process that is constitutionally active in essentially all eukaryotic tissues [ 33 ] and is required to generate new space for newly proliferated cells during unremitting cell generation, in our experiments and probably in mouse taste organs.

It is possible that excessive insulin could interrupt normal cell apoptotic processes and the consequent smooth cell turnover. In this context, insulin could be one of the important regulators to maintain normal taste cell turnover. And a subset of ENaC-expressing salt-responsive taste cells might express IR because amiloride-sensitive salt taste responses were enhanced by insulin via controlling the open probability of ENaC and transport of ENaC proteins to the membrane [ 4 ], [ 5 ].

Altogether, as our work demonstrated, IR expression in taste buds may not be restricted to specific taste cells types, and a large population of taste cells may express IR. According to previous reports, IGF1 receptor IGF1R , which shares a high degree of homology with IR and has low affinity for insulin, is expressed in taste bud cells, and some overlapped with keratin 18 [ 21 ], [ 1 ].

Young days-old but not adult days-old mice genetically lacking IGF1R in lingual epithelium exhibited decreased taste bud numbers [ 21 ]. In addition, IGF1R deletion did not affect taste bud size and taste cell population [ 21 ].

According to results of our qPCR experiments with taste organoids, the taste cell marker krt8 and each cell-type marker NTPDase2, T1R3, gustducin, and CA4 were significantly decreased with higher insulin concentrations Fig 3.

Both IR and IGF1R were shown to have broad expression patterns in taste buds Fig 1B and 1C and [ 1 ] , but their multiple specific functions have yet to be studied in various cell types and organs. Taste disorder has been described frequently during the course of diabetes, for example, increased recognition thresholds for glucose, NaCl [ 34 ], and sucrose [ 35 ] and impaired sweet, sour, and salt taste detection in type II diabetic patients [ 36 ].

Hyperactive mTORC1 has been observed in obesity and nutrient overload, probably due to hyperglycemia and hyperinsulinemia [ 7 ]. According to our results, the hyperinsulinemia that advances with overweight could impact the proliferation of all taste cell types and might be a potential risk factor for impairment of total taste sensitivity.

The ex vivo taste cell culture system revealed that insulin negatively regulated taste cell generation, including type I, II, III, and taste progenitor cells, and pharmacological inhibition of mTOR significantly promoted taste cell proliferation. We thank Dr. Wenwen Ren for advice on culturing taste bud organoids, Dr. Hans Clevers for providing the Wnt3a-producing cell line, Dr. Jeffery Whitsett for the R-spondin-producing cell lines, and Dr.

Patricia J. Watson helped us about English writing and editing. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Abstract Expression of insulin and its receptor IR in rodent taste cells has been proposed, but exactly which types of taste cells express IR and the function of insulin signaling in taste organ have yet to be determined.

Data Availability: All relevant data are within the manuscript. Introduction Insulin is an essential hormone for managing energy within the body. Materials and methods Animals Mouse husbandry and all mouse experiments were carried out under the ethical guidelines of Kyushu University.

Taste bud organoids 3-D taste bud organoids were prepared as previously described [ 25 ]. Download: PPT. Fig 1. Insulin effects on expression of taste cell markers and organoid colony size To investigate the effect of insulin on taste cell growth, we applied various concentrations of insulin to isolated taste progenitor cells. Fig 3. Insulin effects on taste cell type marker mRNA in taste organoids.

Fig 5. The significance of these different morphological cell classifications in terms of function, structure, and lineage are not known because taste cells are continuously renewed from basal cells and may vary with age Beidler and Smallman, ; Farbman, Furthermore, cytochemical signatures vary among species Takeda et al.

It has been hypothesized that formation of taste buds is induced by cranial nerve fibers during embryonic development Fujimoto and Murray, ; Hosley and Oakley, ; Oakley et al. Studies have also shown that denervation of gustatory nerves leads to the disappearance of taste buds Morris-Wiman et al.

Contrary to these neuronal induction theories, other studies have shown that taste cells can differentiate fully without requiring any nerve innervation. The induction of taste cell development may be triggered by signals from other tissues or may arise independently through intrinsic signaling mechanisms that are associated with the epithelium during late embryonic development Barlow and Northcutt, ; Barlow, Individual taste buds arise from multiple progenitors.

However, it is not known if these progenitors give rise to basal cells that generate different taste cell types or to multipotent epithelial stem cells that generate lineage-restricted basal cells Stone et al. Cell culture techniques can be a valuable approach to advance our knowledge and understanding of the molecular structure and cellular physiology of taste cells and the processes of proliferation, differentiation, and regeneration.

Currently, there are no longer term in vitro taste cell models to study function and development because of the limitations of primary cultures and the difficulties in maintaining sensory cells in vitro. These limitations may be caused by taste cell isolation procedures, mechanical stresses, and harsh enzyme treatments, which may degrade structures and reduce cellular viability Spielman et al. Previous studies indicated that maintaining rat primary cells beyond 3—5 days was not possible based on the specified isolation procedures and culture techniques Spielman et al.

We aimed to revisit the problem in view of the established need for such a system. We report for the first time the establishment of an in vitro culture system for the long-term maintenance of isolated rat taste cells by developing isolation methods and selecting culture media and supplements that are essential for cell attachment, growth, and differentiation.

Isolated rat taste cells from circumvallate and foliate papillae were maintained for more than 2 months in culture with minimal loss of viability and which maintained the expression of characteristic molecular and physiological features. Calcium imaging demonstrated that cultured taste cells also responded to different taste stimuli, indicating functional maturation.

Rats ranging from 1 to 2 months old were euthanized by CO 2 inhalation followed by cervical dislocation. The tongue was dissected proximal to circumvallate papillae and immediately placed into an isolation solution [26 mM NaHCO 3 , 2. The preparation was then removed from ice and approximately 1 ml of the isolation buffer mixed with 1.

After 15—20 min of incubation in isolation buffer at room temperature, the epithelium was gently peeled from the underlying muscle layer under a dissecting microscope Stereomaster, Fisher Scientific, Pittsburgh, PA and placed in isolation solution. The pieces were seeded onto mm round glass coverslips Fisher coated with rat tail collagen type 1 3. Culture medium was replaced after 48 h and then every 5—7 days.

In preliminary studies, we also examined different coating materials and medium supplements to determine the optimal culture conditions. We also examined different compositions of tissue culture medium for maintaining taste cells [i. Assessment of cultured taste cell viability was done by staining with 0. Based on cell attachment and viability over 7—10 days in culture observed in these preliminary experiments, we selected the protocol described above for subsequent studies.

Western blots were conducted using standard immunoblotting techniques as previously described Sambrook et al. Cultured primary rat taste cells were lysed, and tissue samples from rat cicumvallate and foliate papillae were homogenized in radioimmunoprecipitation assay RIPA buffer [ mM NaCl, 10 mM Tris pH 7. After 1. Signal was detected with the enhanced chemiluminescence immunoblot detection system Amersham Biosciences following the manufacturer's instructions.

X-ray films were later scanned for documentation and analysis. Epithelial tissues were isolated as described above and homogenized into 1 ml of cold RIPA buffer supplemented with protease inhibitor cocktail.

Homogenate was clarified at rpm for 4 min. The beads were washed three times with 0. Primers were chosen to span one or more introns to exclude confusion with amplified fragments from genomic DNA. BrdU incorporation was used to monitor cell proliferation. Coverslips were thoroughly washed with PBS and water and then mounted onto slides with Vectashield Vector Laboratories.

For immunofluorescence double labeling, the coverslips were reblocked with 0. To determine the specificity of gustducin staining, gustducin antibody preincubated with fivefold by weight of its specific peptide Santa Cruz Biotechnology, sc P for 2. Controls for immunofluorescence consisted of omitting the primary antibody or substituting the primary antibody with the host IgG from which the antibody was generated.

In addition, for double-labeling experiments, controls were done in which either the first or second primary antibody was omitted with all other steps in the protocol maintained to check for nonspecific interactions. In all cases, these controls revealed no artifactual labeling.

Immunoreactive cells were counted in at least three sampling fields. Excitation wavelengths used were at nm for DAPI, nm for Alexa Fluor , and nm for Alexa Fluor with emissions detected at appropriate wavelengths. The pinhole diameter was set at the first minimum diameter of the Airy disc for the objective used, giving acceptable resolution of the z -axis for the fluorescent focal plane.

Sequential acquisition of each wavelength was used for some double-labeling experiments to prevent cross talk or bleed through between fluorophores. Computer-controlled digital zoom was used to increase magnification to a maximum of 2. Coverslips were then placed in a recording chamber and continuously bathed with MHNK solution superfusion.

Cells were exposed to various chemical stimuli: 0. The chemical stimuli were applied to the coverslip by switching the superfusion to the stimulus solution, which allowed for a complete change of bath solutions in the chamber within 10 s. Calcium-imaging recordings were performed using standard imaging techniques Rawson et al. Cells were illuminated with light emitted by a W Xenon lamp alternately filtered with narrow band-pass filters at nm, then nm. Exposure times were minimized and light shuttered between acquisitions to minimize photobleaching.

Images were digitized using a Merlin Imaging Workstation Perkin Elmer Life Sciences , which controlled the illuminator and camera, and acquisition, and performed the image ratioing and display of pseudocolor images.

Cells remained viable in the recording setup for over 2 h without visible effects of dye bleaching. Stimuli were diluted in MHNK buffer and applied via a gravity-flow superfusion apparatus for 10—60 s, depending on the stimulus. Only records in which calcium levels returned to within 0. The method for taste cell isolation was evaluated with respect to enzymatic treatment for cell isolation, coating of the cell culture plates and coverslips, and composition of tissue culture medium.

The isolation procedure was rendered more efficient by using a single isolation solution with a shorter incubation time than described in previous studies and utilizing two protein digestion enzymes, which required less treatment time. Culturing rat taste cells with UV-irradiated mouse fibroblast cells as cofeeder had technical and experimental limitations since mouse fibroblast cells became detached from tissue culture plates Figure 1B. No cell attachment was observed on the surface of uncoated tissue culture plates polyprene and glass coverslips coated with Figure 1D or without poly- D -lysine.