Small Volume Of Medium Research Articles

A novel hot-tamping process for producing an improved quality of coke

To produce superior quality of coke as compared to traditional cokemaking technologies, a novel hot-tamping technique has been developed. The quality of produced coke was assessed based on the reactivity, pore structure and microcrystalline structure. The results confirmed that the hot tamping coking process produced cokes with superior qualities as compared to traditional cokemaking and cold tamping coking. The hot-tamping cokes have good gloss, compact carbon structure, strong thermal strength and small volume of medium and small pores. This will reduce the coke reactivity by 3–4% when compared with traditional cokemaking process. The hot tamping cokes have the smallest spacing of the crystal face layers with the highest stacking height, diameter, crystal surface size, and graphitization degree that was hardly observed for the cokes from traditional cokemaking and cold tamping coking.

Study of a Cold Atmospheric Pressure Plasma jet device for indirect treatment of Squamous Cell Carcinoma

PurposeThe possibility of obtaining plasmas at low temperatures and atmospheric conditions, the Cold Atmospheric Plasmas (CAPs), has triggered several studies on its possible medical applications. The use of CAPs devices applied to oncological treatments is still in a pre-clinical stage, but early investigations have shown promising results, both in vitro and in vivo. In this work, is reported the study of a custom made Cold Atmospheric Pressure Plasma jet device for indirect treatment of Squamous Cell Carcinoma. MethodsThe plasma plume produced by the jet device was characterized by optical emission spectroscopy and the vulnerability of two different cell lines, SCC-15 (Squamous Cell Carcinoma) and HGF-1 (non-cancer cell line) to different working parameters factors such as the volume of medium used on indirect plasma treatments, the number of cells used in the biological assays and the volume of treated medium in contact with the cultured cells were systematically tested and studied. ResultsThe effectiveness of CAPs’ treatments is time, volume and cell dependent. Cancer cells, specifically SCC-15 cells have shown to be more sensitive to indirect plasma treatments than the non-cancer cell line used in the present work. ConclusionsAccording to the obtained results, this study showed that thein vitro anti-cancer capacity of the developed jet plasma device will be higher when small volumes of medium are used together with moderate plasma treatment times, keeping the non-cancerous cells almost unaffected.

Uniform, fast, high concentration delivery of bone marrow stromal cells and gingival fibroblasts by gas-brushing

Delivering multipotent stem cells in adequate milieu directly to site of injury is important for successful tissue regeneration. Accelerated wound healing has been reported in recent publications when large area wounds were treated with sprayed cells. However, little is known about the basic physics of cell spraying in hydrogel free medium, causes of concomitant cell damage, and whether it can be applied for craniofacial tissue regeneration. To answer some of these questions, a simple co-axial CO2 gas operated brush (g-brush) was used to deliver human bone marrow stromal (hBMSC) and mouse gingival fibroblast (ESK-1) cells at low gas flow rates and in a small volume of medium. Live/dead, metabolic activity (MTS-1) and total DNA quantification assays were utilized to evaluate cell cultures after g-brush and typical pipette deposition. Viable hBMSC and ESK-1 cells were uniformly delivered at higher cell concentrations (≥60 000), in a short period of time (15 and 9 s), and in a small fluid volume (0.25 ml). Significantly, after the hBMSC were g-brushed and cultured up to 21 d, the cell multipotency was found to be preserved and comparable to the pipetted cells. Fluid dynamic calculations highlighted importance of low interfacial shear stress (<35 Pa) and gas velocity (<160 m s−1) in delivering concentrated, viable cells. Results of this research support applicability of the g-brushing technique for the quick, large number delivery of viable cells for tissue regeneration applications.

Development of a New Method for Bioassay of Allelopathy Using Protoplasts of a Leguminous Plant Mucuna pruriens With a High Content of the Allelochemical L-DOPA

To study allelopathy at the cellular level, we established a new bioassay method using protoplasts from etiolated leaves of a leguminous plant, Mucuna pruriens, which contains a large amount of L-3,4-dihydroxyphenylalanine (L-DOPA) and protoplasts prepared from cotyledons, leaves or roots of Lactuca sativa (lettuce) grown in an axenic condition. The bioassay was performed using 96 multi-well culture plates and required only a small volume of medium, 50 ?L, per well. For the mixed culture of Mucuna and recipient lettuce protoplast, the optimum condition was Murashige and Skoog’s basal medium, containing 1 ?M of 2,4-dichlorophenoxyacetic acid, 5 ?M of benzyladenine and 0.4 M mannitol solution. The inhibitory effect of M. pruriens protoplasts was found to vary with the density of the recipient L. sativa protoplasts. The M. pruriens protoplasts had no effect on the division of protoplasts from suspension cells of Oryza sativa. These results were in agreement with those obtained by the conventional plant box method. Our study offered a novel in vitro assay method, which will be widely applicable to study allelopathy of various plants on lettuce and also for quantitative studies between plant species under different culture conditions in order to simulate the possible future environmental risk.

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in which the enhanced version of the green fluorescent protein (EGFP) is exclusively expressed in all neurons of the developing central and peripheral nervous system1, allowing the possibility to both film the innervation of the forelimb and to manipulate this process with pharmacological and genetic techniques2. The most critical parameter in the successful cultivation of such slice cultures is the method by which the slices are prepared. After extensive testing of a variety of methods, we have found that a vibratome is the best possible device to slice the embryos such that they routinely result in a culture that demonstrates viability over a period of several days, and most importantly, develops in an age-specific manner. For mid-gestation embryos, this includes the normal outgrowth of spinal nerves from the spinal cord and the dorsal root ganglia to their targets in the periphery and the proper determination of skeletal and muscle tissue. In this work, we present a method for processing whole embryos of embryonic day (E) E10 to E12 into 300 - 400 micrometer slices for cultivation in a standard tissue culture incubator, which can be studied for up to two days after slice preparation. Critical for the success of this approach is the use of a vibratome to slice each agarose-embedded embryo. This is followed by the cultivation of the slices upon Millicell culture membrane inserts placed upon a small volume of medium, resulting in an interface culture technique. One litter with an average of 7 embryos routinely produces at least 14 slices (2-3 slices of the forelimb region per embryo), which varies slightly due to the age of the embryos as well as to the thickness of the slices. About 80% of the cultured slices show nerve outgrowth, which can be measured througout the culturing period2. Representative results using the tauGFP mouse line are demonstrated.

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in which the enhanced version of the green fluorescent protein (EGFP) is exclusively expressed in all neurons of the developing central and peripheral nervous system(1), allowing the possibility to both film the innervation of the forelimb and to manipulate this process with pharmacological and genetic techniques(2). The most critical parameter in the successful cultivation of such slice cultures is the method by which the slices are prepared. After extensive testing of a variety of methods, we have found that a vibratome is the best possible device to slice the embryos such that they routinely result in a culture that demonstrates viability over a period of several days, and most importantly, develops in an age-specific manner. For mid-gestation embryos, this includes the normal outgrowth of spinal nerves from the spinal cord and the dorsal root ganglia to their targets in the periphery and the proper determination of skeletal and muscle tissue. In this work, we present a method for processing whole embryos of embryonic day (E) E10 to E12 into 300 - 400 micrometer slices for cultivation in a standard tissue culture incubator, which can be studied for up to two days after slice preparation. Critical for the success of this approach is the use of a vibratome to slice each agarose-embedded embryo. This is followed by the cultivation of the slices upon Millicell culture membrane inserts placed upon a small volume of medium, resulting in an interface culture technique. One litter with an average of 7 embryos routinely produces at least 14 slices (2-3 slices of the forelimb region per embryo), which varies slightly due to the age of the embryos as well as to the thickness of the slices. About 80% of the cultured slices show nerve outgrowth, which can be measured througout the culturing period(2). Representative results using the tauGFP mouse line are demonstrated.

Pectin-Based Bioadhesive Delivery of Carbenoxolone Sodium for Aphthous Ulcers in Oral Cavity

The objective of this study was to prepare and evaluate the pectin-based dosage form for buccal adhesion. Carbenoxolone sodium, which is used for the treatment of aphthous ulcers in oral cavity, was used as a model drug. The pectin buccal discs were prepared by direct compression. The water uptake and erosion of pectin disc increased progressively with the swelling time. The bioadhesion of dried pectin discs decreased when either the discs were hydrated or the buccal tissue was wet with a small volume of medium. The influencing factors such as pectin type, pectin to lactose ratio, and sweetener type on the formulations were investigated. The results demonstrated that buccal discs prepared from pectin with a high degree of esterification (DE) showed a weaker and more friable characteristic than that with low DE. Decreasing pectin to lactose ratio resulted in the high dissolution rate with low bioadhesive properties. Addition of sweetener in the formulations also affected the hardness, friability, and bioadhesive properties of the discs. The pectin discs containing sweetening agent showed a higher drug release than those without sweetener. The results suggested that pectin-based bioadhesive discs could be used to deliver carbenoxolone sodium in oral cavity.

174 TRANSCERVICAL EMBRYO COLLECTION IN THE DOMESTIC BITCH

Embryo collection in the bitch is usually performed via surgical collection or uterine excision. Our objective was to collect embryos nonsurgically, or with minimal surgical invasion. Estrus was monitored using vaginal cytology and progesterone analysis during 1 natural cycle and 13 cycles induced by the submucosal insertion of a 2.1-mg deslorelin implant into the vestibulum of Walker-type hounds (n = 10, 30 kg). Bitches (n = 14 cycles) were transcervically inseminated twice in 12 cycles, (3.3 and 5.2 days post-LH) and once on 2 cycles (Day 6 post-LH) with fresh semen. Uterine flushes (n = 14) using an equine medium (ViGro, Bioniche Animal Health USA Inc., Athens, GA, USA) were done 13 to 15 days after the LH peak. Nonsurgical, nonanesthetized flushes were attempted in 9 cycles. In one cycle an 8-Fr, 55-cm Foley catheter with a 1-mL cuff (SurgiVet, Waukesha, WI, USA) stiffened with a metal stylette was inserted through the cervix using a 22-Fr sheath (63027KL, Karl Storz Veterinary Endoscopy, Goleta, CA, USA) and a 3.5-mm cystoscope, (63325BA, Karl Storz Veterinary Endoscopy) and flushed with 1.5 mL of medium infused and recovered by aspiration. In a one bitch, an 8-Fr polypropylene catheter (Sovereign, Tyco Healthcare, Mansfield, MA, USA) was transcervically passed after failing to pass an 8-Fr Foley catheter, but a flush was not done. In 5 cycles, the 22-Fr sheath could not be passed into the cranial vagina, so a 9.5-Fr, 43-cm cystoscope (27012L, Karl Storz Veterinary Endoscopy) was used to transcervically pass a 5-Fr, non-cuffed catheter (17500/0005, Minitube of America, Verona, WI, USA). Only non-cuffed catheters could be passed through the 9.5-Fr cystoscope, so no flushes were attempted. In 2 cycles, the cervix could not be visualized. Surgical flushes under anesthesia were attempted in 5 cycles. One was by transcervical catheterization using an 8-Fr catheter identical to the nonsurgical attempts, but adding surgical exteriorization of the uterine horns and retrograde flushing with 20 mL of medium. In 4 bitches the uterus was exteriorized by ventral midline laparotomy; one had a pyometra at surgery, and in three, 14-gauge i.v. catheters (Angiocath, BD, Sandy, UT, USA) were placed in the tip and body of each horn, and the uterus was retrograde flushed with 20 mL of medium. No embryos were recovered by the nonsurgical and/or transcervical flushes. Passing the 22-Fr sheathed cystoscope that accommodated a cuffed catheter into the cranial vagina, passing a cuffed catheter, or visualizing the cervix was unsuccessful in 7 of 9 cycles. Uterine distension appeared to cause discomfort during nonsurgical flushes. Three of 5 bitches that had a 5-Fr, non-cuffed catheter were subsequently pregnant. One blastocyst was recovered after surgical flushing with the i.v. catheters in the uterus. Nonsurgical embryo recovery in the bitch was most likely unsuccessful, compared to ours and previous work, due to inabilities to pass a cystoscope into the cranial vagina, to pass a catheter through the cervix, to visualize the cervix, or to create uterine distension using a small volume of medium. Pregnancies were maintained after successful transcervical catheterization of mated bitches.

Fabrication and characterization of poly(lactic-co-glycolic acid)/polyvinyl alcohol blended hollow fibre membranes for tissue engineering applications

Hollow fibre membrane bioreactors are ideal for tissue engineering applications because they can offer a large surface area to volume ratio in order to culture large numbers of cells in relatively small volumes of media. Poly(lactic-co-glycolic acid) (PLGA) hollow fibres have great potential for use in tissue engineering due to their biocompatibility and biodegradability, but their hydrophobicity results in low hydraulic permeability and limited cell adhesion. To overcome this, PLGA hollow fibres have been previously used immediately after fabrication, while still wet, or have been dried for storage and subsequently wetted with ethanol. These methods both present problems since the fibres cannot be stored wet as they degrade, while ethanol causes deformation such that they distort in the bioreactor and stick together. Here we present an alternative method to allow wetting of dried fibres by water alone: the addition of polyvinyl alcohol (PVA) to the spinning dope. Blending of PLGA and PVA resulted in a concentration-dependent increase of the mean pore size, porosity, contact angle, and a corresponding decrease in mechanical strength. To determine whether membranes were amenable to transport of culture medium and its constituents, permeation of water and a model protein, BSA, was assessed. No pure water or protein permeation was observed in PLGA hollow fibre membranes that had been dried for storage or PVA–PLGA blends with 1.25 and 2.5% PVA. However, fibres with 5% PVA that had been dried for storage allowed permeation of both water and BSA, without rejection of, or fouling by the protein. It was concluded that 5% PVA was sufficient to obtain wetting with water alone, and transport properties suitable for the permeation of media proteins without a significant or detrimental effect on mechanical strength. Therefore a PVA–PLGA blend by the addition of 5% (w/w) PVA to 20% (w/w) PLGA in N-methyl-2-pyrrolidone spinning dope is recommended when preparing PLGA hollow fibre membrane scaffolds.

The effects of time of first cleavage, developmental stage, and delipidation of nuclear-transferred porcine blastocysts on survival following vitrification

The effect of removing cytoplasmic lipid droplets (delipidation) at the 2-cell and developmental stages on the survival of porcine somatic cell nuclear-transferred blastocysts developed from the enucleated oocytes receiving somatic cells from kidney of an adult female after cryopreservation was examined. Vitrification was performed using the Cryoloop method with a small volume of medium (0.5 μl). To select 2-cell embryos with a high potential to develop into blastocysts, the relationship between the timing of the first cleavage and the developmental potential was examined. The potential of nuclear-transferred oocytes to develop into blastocysts in the intermediate-cleavage group (20–24 h after activation, 25%) was slightly or significantly ( P < 0.05) higher than that in fast-cleavage (<20 h after activation, 13%) and slow-cleavage groups (>24 h after activation, 5%). Most non-delipidated blastocysts did not survive after thawing (0% for early-stage and 9% for advanced-stage blastocysts), but the survival rate of delipidated blastocysts 48 h after culture (54% and 72%, respectively) was not significantly different from that of non-vitrified blastocysts (80% and 92%, respectively). The survival rate of advanced-stage blastocysts after vitrification was slightly higher than that of early-stage blastocysts. The present study demonstrates that somatic cell nuclear-transferred porcine blastocysts developed from embryos selected at the 2-cell stage can be preserved by vitrification with a small volume of medium if the lipid droplets of the embryos are first removed.

Growth and isotopic labelling of adherent cells in small volumes of medium

The in vivo labelling of viral or cellular components is usually conducted through the addition of radioactively labelled precursors to the culture medium. A limiting factor for isotope use is often the cost of isotope purchase and disposal. Therefore, significant savings can be achieved if the smallest possible volume of medium is employed. However, in the case of adherent cells growing in tissue culture dishes or multi-well plates, surface tension causes a very uneven distribution of the liquid due to the formation of a meniscus at the edge of the petri. This prevents the use of very small volumes of medium for cell growth and labelling because the cells at the center of the petri dish would dry out and die, especially after longer incubation periods. In this communication, we describe a technique whereby cells are grown in an area surrounded by a hydrophobic ring of Teflon, which greatly improves the distribution of the medium by eliminating the concave meniscus. This translates into a dramatic improvement in the condition of the cells, as well as the efficiency of labelling of phosphoproteins, such as the Simian Virus 40 large tumor antigen with 32P-orthophosphate or labelling of the cellular DNA with 3[H]thymidine. The technique is useful for any application where growth of cells in small volumes of medium is required.

Quantitative analysis of cell detachment by shear stress

It is important to enhance adhesive force of cells to scaffolds in a field of tissue engineering. In this study, we developed a new system that can evaluate detachment force of cells with ease. A cone and plate-type viscometer provides a well-defined homogeneous laminar flow and consists of simple structure. Therefore, the system was based on a modified cone and plate-type viscometer equipped with an upright epifluorescence microscope. The fixation of diverse biomaterials, i.e. plate-, membranous- and porous-shaped materials to the system was enabled possible. In addition, small volume of medium, small number of cells, and small piece of material were enough to evaluate cell detachment by shear stress. By using the newly developed apparatus, the phenomena of cell detachment were quantitatively analyzed with ease. Therefore, our apparatus should be a useful instrument to develop and evaluate the materials that are expected to enhance endothelialization.

Effects of hypoxia and glutathione depletion on hemoglobin- and myoglobin-mediated oxidative stress toward endothelium

We investigated the toxicity of hemoglobin/myoglobin on endothelial cells under oxidative stress conditions that include cellular hypoxia and reduced antioxidant capacity. Bovine aorta endothelial cells (BAECs), grown on microcarrier beads, were subjected to cycles of hypoxia and reoxygenation in a small volume of medium, and endothelial cell monolayers were depleted of their intracellular glutathione (GSH) by treatment with buthionine sulfoximine. Incubation of diaspirin cross-linked hemoglobin (DBBF-Hb) or horse skeletal myoglobin (Mb) with BAECs subjected to 3 h of hypoxia caused transient oxidation of the hemoproteins to the ferryl form (Fe 4+). Formation of the ferryl intermediate was decreased in a concentration-dependent manner by the addition of L-arginine, a substrate of NO synthase, after 3 h of hypoxia. Optimal inhibition of ferryl formation, possibly due to the antioxidant action of NO, was achieved with 900 μM L-arginine. Addition of hydrogen peroxide to GSH-depleted cells in the presence of DBBF-Hb or Mb significantly decreased cell viability. Ferryl Mb, but not ferryl DBBF-Hb, was observed in samples analyzed at the end of treatment, which may explain the greater toxicity observed with Mb as opposed to DBBF-Hb. This model may be utilized to identify causative agent(s) associated with hemoprotein cytotoxicity and in designing strategies to suppress or control heme-mediated injury under physiologically relevant conditions.

A Simple Procedure for in Situ Immunolabeling, Embedding and Sectioning of Layers of Cultured Endothelial and Smooth Muscle Cells for both Light and Electron Microscopy

We present a simple procedure for in situ immunolabeling, embedding and sectioning of layers of cultured endothelial and smooth muscle cells for both light and electron microscopy. Endothelial and smooth muscle cells were seeded in tissue culture chambers /slides precoated with 30% (w/v) gelatin drops fixed with 0.5% glutaraldehyde. Live endothelial cell layers were labeled with an antibody against the surface membrane protein, anti-CD 13. After labeling, the cell layers were fixed and separated from the chambers/slides by lifting all of the samples with a spatula. Sections (1-2 mm) were cut, embedded and processed further for light or electron microscopy. Because of the delicate cell layers and the importance of preserving maximum integrity, labeling was performed under standard culture conditions and treated in situ during the entire procedure. Moreover, the small chamber size of the tissue culture dishes generated the additional advantages of requiring only a limited number of cells, small volumes of media, and little antibody.

A novel technique to overcome browning in tissue culture

Experiments conducted using Dioscorea alata L. revealed that an exudate from the cut end of the explants was responsible for browning of the culture medium. Browning did not affect growth of roots and shoots when explants were cultured in a large volume of medium, but in a small volume it was lethal. Sealing the cut ends with paraffin wax was found to control browning by preventing exudation. This simple technique permitted establishment of cultures in a small volume of medium in about 90 percent of the cases, while in unsealed cultures lethal browning was recorded in 80 percent of the cases. The advantages of this technique over other methods of controlling browning are discussed.

Morphologic characterization of osteoblast-like cell cultures isolated from newborn rat calvaria

Two methods for harvesting osteoblast-like cell populations from newborn (10 days) rat calvaria were compared. The first one consisted in culturing the periosteum-free bones and then trypsinizing the cells on the bone surface. The second one involved the migration of the osteoblasts on glass fragments before trypsinization. Since the plating efficiency, the proportion of alkaline phosphatase-positive cells, the population doubling time, and the calcium deposition were more adequate, the second method was used to further characterize the behavior of the cultures. During the first week of culture, the cells featured shapes similar to those observed in vivo on the surface of periosteum-free calvaria. They formed multilayers and, in the presence of ascorbic acid, synthetized an organic matrix containing exclusively type I collagen. Later, small amounts of type III collagen appeared. The cells were embedded in the matrix and progressively acquired the morphologic phenotype of osteocyte-like cells. The matrix mineralized in the presence of beta-glycerophosphate. The technique of drop-inoculation (high concentration of cells in a small volume of medium) promoted the multilayer formation and the achievement of large mineralized plates (about 1 cm2) in 3 weeks of culture.

Computed Tomographic Analyses of Water Distribution in Three Porous Foam Media

AbstractComputer Assisted Tomography (CAT) is commonly used in diagnostic radiology to make nondestructive images and analyses of cross sections of the human body. CAT scanning may also be useful in imaging and measuring spatial distribution and changes in water distribution in porous media. The purpose of this paper is to review some of the details of CAT scanning that are of importance to the application of CAT scanning porous media and to evaluate the use of the CAT scanner to measure the spatial distribution of water in three different porous media. The scanner's response to changes in the spatial distribution of water in three different porous phenolic foam materials after draining for 16 h was investigated. Water content distributions were successfully detected with good resolution on the x‐ray image. Comparisons of CAT vs. gravimetrically determined water content indicated a significant linear relationship between the methods. Results from these experiments indicate that the CAT scanner can nondestructively measure volume wetness in phenolic foam media. The clarity of the CAT images suggests that CAT scanning has great potential for studies where small and rapid changes in water content within small volumes of media are desired.

Rapid optimization of immunoadsorbent characteristics.

Immunoaffinity chromatography is employed in many research areas. We have developed an assay system that overcomes some of the tediousness and uncertainty in dealing with immunoadsorbents (IA). The preparation of IA and the effect of various procedures on the dissociation of antigen-antibody complex and the regeneration of IA are rapidly screened by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. Non-covalently bound proteins are dissociated and separated during the electrophoresis from IA beads. A wide variety of associative and dissociative conditions can be tested on small amounts of IA. Information about biospecifially and non-biospecifically adsorbed proteins can also be obtained. By using relatively small volumes of media containing either an antigen or another biospecific molecule, the optimal parameters for affinity chromatography (specificity, binding capacity, efficiency of solvents in dissociation of the complex and their effect on the adsorbent), or even for ion-exchange chromatography, can be determined without first performing several time- and material-consuming chromatographic experiments.

Bidirectional movement of gamma-aminobutyric acid in rat spinal cord slices.

The bidirectional movement of GABA (gamma-aminobutyric acid) was studied in slices of rat spinal cord which were incubated in small volumes of medium. The appearance in the medium of endogenous GABA and the disappearance from the medium of [14C]GABA were used to calculate the rates of unidirectional uptake and unidirectional release of GABA. Under these conditions, no net uptake of GABA was observed when slices were incubated in media containing concentrations of gABA as high as 25 microM. Elevated potassium (60 mM) stimulated the unidirectional release of endogenous GABA from spinal cord slices by a calcium-dependent process. Ouabain (0.1 mM) more than doubled the unidirectional release of endogenous GABA in a calcium-independent manner, while unidirectional uptake was inhibited by 44%. Nipecotic acid (1.0 mM) stimulated unidirectional release and inhibited unidirectional uptake of GABA.

Bidirectional movement of γ-aminobutyric acid in cerebral cortex slices incubated in small volumes of medium

Rat cerebral cortex slices were incubated in small volumes of medium in order to study the bidirectional movement of γ-aminobutyric acid (GABA) between tissue and medium, and to measure the extent of release of endogenous amino acids of interest as potential neurotransmitters. The apparent uptake, real uptake, and net change of GABA were calculated from the changes in [ 14C]GABA and endogenous GABA in the medium. The extent of release of endogenous amino acids was dependent upon the amino acid, the duration of incubation, and the volume of incubating medium. Substantial amounts of endogenous amino acids may accumulate in the medium under conditions commonly employed in amino acid uptake studies. The accumulation of endogenous GABA may affect the calculation of GABA uptake when this is based on the initial specific activity of exogenous [ 14C]GABA. The difference between apparent uptake and real uptake was particularly prominent in the presence of the depolarizing agents, potassium or veratridine, which caused a marked increase in GABA release. Exchange or net release of GABA was found when slices were incubated briefly in media containing 1.3 μM or 12.5 μM exogenous GABA; net uptake accounted for 50% of observed uptake when slices were incubated in media containing 25 μM GABA.