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PubMed1994

Measurement of hybridoma cell number, viability, and morphology using fully automated image analysis.

A novel automated image analysis method is described for characterizing the viability and morphology of animal cells from suspension cultures. With the aid of the exclusion dye Trypan Blue, the total and viable cell counts and the percentage of dead cells present are found. The area, perimeter, equivalent diameter, and circularity of the projected image of each cell are also measured, allowing the estimation of cell volume. The image analysis method is inherently sensitive, precise, consistent (non-operator dependent) and relatively fast, taking approximately 22 min to analyze one sample. The data it gives on individual cellular activity, as characterized by Trypan Blue uptake and cell morphology, are valuable in allowing early diagnosis of even subtle changes in the health of a culture. The method should permit better optimization of culture conditions, and will provide data for the modeling of cell population dynamics. As the conventional manual method is operator dependent, relatively limited in the amount of information it provides, and has a tendency to underestimate Trypan Blue takeup, it is suggested that image analysis be the preferred option for animal cell counting and viability determinations.

Animals↗
PubMed1996

Extending the margin of safety of preservation period for resuscitation of ischemically damaged pancreas during preservation using the two-layer (University of Wisconsin solution/perfluorochemical) method at 20 degrees C with thromboxane A2 synthesis inhibitor OKY046.

We have shown that 5-hr preservation using the two-layer (University of Wisconsin solution/perfluorochemical) method at 20 degrees C allows ATP synthesis and makes it possible to resuscitate a canine pancreas subjected to 90 min of warm ischemia. However, 8 hr of preservation using this method caused a disturbance of vascular microcirculation and did not resuscitate the grafts. The aim of this study was to examine the effect of thromboxane A2 synthesis inhibitor OKY046 on vascular endothelial cells and ATP tissue levels of canine pancreas during preservation using the two-layer (University of Wisconsin solution/perfluorochemical) method at 20 degrees C, and vascular microcirculation and pancreas viability after transplantation. Graft viability was judged by graft survival following autotransplantation. ATP tissue levels were measured by high-performance liquid chromatography at the end of preservation. Viability of the vascular endothelial cells was judged using nuclear trypan blue uptake of the graft after preservation. Pancreatic tissue perfusion was measured using an H2 clearance technique after reperfusion. Pancreas grafts subjected to 90 min of warm ischemia were not viable (0/5). However, 5-hr preservation made it possible to recover the pancreas (5/5); 8-hr preservation was not successful (0/3). ATP tissue levels after 5-hr and 8-hr preservation were 9.40+/-2.09 and 7.37+/-1.06 micromol/g dry weight, respectively, and OKY046 did not affect ATP synthesis during 8-hr preservation (8.44+/-0.92 micromol/g dry weight). The percentage of nuclear trypan blue uptake of endothelial cells in 8-hr-preserved grafts was 37.6+/-11.6% and was significantly higher than the value in 5-hr-preserved grafts (5.0+/-3.0%; P<0.01). However, OKY046 significantly reduced trypan blue uptake in 8-hr-preserved grafts (8.2+/-3.6%; P<0.01). Pancreatic tissue perfusion in 8-hr-preserved grafts after 2 hr of reperfusion was 28.5+/-7.5 ml/min/100 g, and was significantly lower than the value in 5-hr-preserved grafts (57.1+/-4.4 ml/ min/100 g; P<0.01), but OKY046 dramatically improved pancreatic tissue perfusion (97.1+/-14.6 ml/min/100 g; P<0.01). As a consequence, 8-hr-preserved grafts were resuscitated (4/5). We conclude that OKY046 protects the vascular endothelium during preservation by the two-layer method at 20 degrees C and consequently improves vascular microcirculation on reperfusion. Together with ATP synthesis, which is essential for repairing damaged cells, the canine pancreas graft subjected to 90 min of warm ischemia is resuscitated during 8-hr preservation by the two-layer method at 20 degrees C. This method holds promise for pancreas-kidney transplantation from cardiac arrest donors.

Adenosine↗
PubMed2004

Evaluation of the direct toxicity of trioctyltrimellitate (TOTM), di(2-ethylhexyl) phthalate (DEHP) and their hydrolysis products on isolated rat hepatocytes.

Plasticizers are added to polyvinyl chloride (PVC) to confer flexibility to the polymer. Di(2-ethylhexyl) phthalate (DEHP) is the most commonly used of them. However, due to its non covalent bond to the PVC, DEHP tends to vaporize easily. A significant exposure has been recorded in dialyzed patients since medical tubings. Most animal species metabolize DEHP rapidly into monoethylhexyl phthalate (MEHP) and 2-ethylhexanol (2-EH). Because of the suspected toxicity of DEHP, an alternative plasticizer, trioctyltrimellitate (TOTM) has aroused increasing interest. The aim of this study was to determine on isolated rat hepatocytes in vitro, the direct hepatotoxic potential of both DEHP and TOTM and their hydrolytic products. To evaluate the possible toxic liver risk resulting from exposure to DEHP and TOTM, isolated rat hepatocytes were incubated with either DEHP, TOTM, MEHP or their common metabolite (2-EH) for 3 hours. Cell viability was periodically estimated thanks to trypan blue tests (15 - 180 min). The activity of lactate dehydrogenase (LDH) was also monitored (1h, 2h, 3h). The results obtained with trypan blue test and with direct LDH activity measurements, were satisfactorily correlated. Hepatocytes treated with both plasticizers and metabolites on the one hand, and the controls (untreated suspension) on the other hand, showed important differences as for cell viability. The acute toxicity on hepatocytes is mainly due to MEHP. Among DEHP, TOTM, MEHP, 2-EH and after intraperitoneal injection of those compounds, only DEHP and MEHP were able to induce a significant hydrogen peroxide (H2O2) production by the rat hepatocytes. These observations enable us to confirm the hypothesis according to which DEHP and MEHP cause an imbalance between the synthesis and the degradation of H2O2. Our results suggest a short-term in vitro cytotoxicity of MEHP. Even if trypan blue and LDH tests offered good results and were easily branded, further assays as well as MTT-tests should performed in order to confirm the cytotoxicity of the compounds tested.

Animals↗
PubMed1988

Maintenance of integrity, viability, and adhesion of Entamoeba histolytica trophozoites in different incubation media.

We have determined the integrity, viability and adhesion of Entamoeba histolytica HK9 and HM1 trophozoites during their incubation in two basal culture media (TP and TYI) and three saline media ("maintenance medium" MM-1 and two others buffered with HEPES). In basal culture media, more than 70% of the trophozoites maintained their integrity and adhesion to human red blood cells (RBC) for up to 4 h, and the proportion of those excluding Trypan blue decreased slowly after 2 h. In saline media, the number of ameba-RBC complexes reached a maximum after 20-30 min and then decreased rapidly (and fastest in MM-1), less than 10% of the amebae were intact after 3-4 h, and dye exclusion fell abruptly from the start of incubation. The number of ameba-RBC complexes formed and the rate of adhesion were highest in basal TP medium. Normal nonvacuolated refringent (NVR) trophozoites deteriorated progressively in all media--although much faster in the saline ones--to vacuolated refringent (VR), nonrefringent, and disrupted. Trypan blue was excluded by all NVR and a fraction of the VR trophozoites. Horse serum helped to maintain ameba integrity and viability, but inhibited adhesion in a concentration-dependent manner. We conclude that E. histolytica trophozoite integrity and adhesion are adequately preserved and should be characterized only in basal culture media, that refringence without vacuolization is a more stringent characteristic of ameba quality than Trypan blue exclusion, and that some serum component inhibits ameba adhesion.

Animals↗
PubMed2002

Different patterns of renal cell killing after warm and cold ischemia.

Kidneys preserved for transplantation surgery sustain injuries caused by cold ischemia during storage. Additionally, kidneys harvested from non-heart-beating donors encounter the stress of warm ischemia. The aim of this study was to determine the specific cell types losing viability after warm and cold ischemia. In warm ischemia studies, the pedicles of left kidneys of Lewis rats were cross-clamped for up to 90 min. In cold ischemia studies, kidneys were flushed with cold University of Wisconsin solution and stored up to 48h at 0-1 degrees C. After warm or cold ischemia, kidneys were perfused via the renal arteries with Krebs-Henseleit bicarbonate (KHB) buffer at 37 degrees C, followed by trypan blue to label the nuclei of nonviable cells. Warm ischemia for 90 min caused renal failure and led to injury of proximal tubular cells, e.g., loss of brush borders, cast formation and trypan blue labeling. Cold ischemia for 48 h also caused renal failure but, unlike warm ischemia, caused trypan blue labeling of glomerular podocytes and peritubular endothelial cells. In warm ischemia-induced injury, electron microscopy showed shedding of microvilli and marked swelling of proximal tubular cells, microvilli and mitochondria. In cold ischemia-induced injury, podocytes were blebbed and swollen, and their pedicels were detached from the basement membrane, but disruption in proximal tubules was milder. In conclusion, warm ischemia triggers injury primarily to proximal tubular cells, whereas cold ischemia damages glomerular podocytes and peritubular endothelial cells in addition to proximal tubules.

Animals↗
PubMed2003

Assessment of cell concentration and viability of isolated hepatocytes using flow cytometry.

The assessment of cell concentration and viability of freshly isolated hepatocyte preparations has been traditionally performed using manual counting with a Neubauer counting chamber and staining for trypan blue exclusion. Despite the simple and rapid nature of this assessment, concerns about the accuracy of these methods exist. Simple flow cytometry techniques which determine cell concentration and viability are available yet surprisingly have not been extensively used or validated with isolated hepatocyte preparations. We therefore investigated the use of flow cytometry using TRUCOUNT Tubes and propidium iodide staining to measure cell concentration and viability of isolated rat hepatocytes in suspension. Analysis using TRUCOUNT Tubes provided more accurate and reproducible measurement of cell concentration than manual cell counting. Hepatocyte viability, assessed using propidium iodide, correlated more closely than did trypan blue exclusion with all indicators of hepatocyte integrity and function measured (lactate dehydrogenase leakage, cytochrome p450 content, cellular ATP concentration, ammonia and lactate removal, urea and albumin synthesis). We conclude that flow cytometry techniques can be used to measure cell concentration and viability of isolated hepatocyte preparations. The techniques are simple, rapid, and more accurate than manual cell counting and trypan blue staining and the results are not affected by protein-containing media.

Adenosine Triphosphate↗
PubMed2005

Staining properties of deepithelialized human amniotic membrane.

PURPOSE: The surgical use of human amniotic membrane (HAM) can be challenging because of its transparency, thin structure, and adhesive quality. Staining of HAM has been proposed to facilitate intraoperative visualization. We evaluated the in vitro staining properties of deepithelialized HAM using 5 vital dyes. METHODS: Deepithelialized HAM was stained with indocyanine green (1.0%, 0.5%, 0.1%), fluorescein sodium (1.0%, 0.25%, 0.1%), lissamine green (0.5%, 0.1%), rose bengal (1.0%, 0.1%), and trypan blue (0.5%, 0.1%). The staining of each dyed sample was evaluated using a x10 operating microscope by a single observer. Each membrane was then rinsed, resuspended (3 mL of balanced salt solution), and then reevaluated for staining at 30-minute intervals for a total of 4 hours. All remaining stained samples were then placed in 5 mL balanced salt solution and then reevaluated for staining at 24 hours. RESULTS: All concentrations of the 5 dyes stained the membrane initially. After 120 minutes, fluorescein sodium 0.1% and lissamine green 0.5% and 0.1% no longer stained the membrane. Fluorescein sodium 0.5% no longer stained at 210 minutes, and fluorescein sodium 1.0% no longer stained at 24 hours. All concentrations of indocyanine green, rose bengal, and trypan blue stained positively at 24 hours. CONCLUSIONS: All 5 dyes stain deepithelialized HAM initially. Tested concentrations of fluorescein sodium and lissamine green may be superior to other tested dyes for intraoperative use because these dyes stain the membrane and then fade. Tested concentrations of rose bengal, trypan blue, and ICG may not be ideal for clinical use because they demonstrate persistent staining at 24 hours.

Amnion↗
PubMed2006

Assembled microneedle arrays enhance the transport of compounds varying over a large range of molecular weight across human dermatomed skin.

In this study, we demonstrate the feasibility to use microneedle arrays manufactured from commercially available 30G hypodermal needles to enhance the transport of compounds up to a molecular weight of 72 kDa. Piercing of human dermatomed skin with microneedle arrays was studied by Trypan Blue staining on the SC side of the skin and transepidermal water loss measurements (TEWL). Passive transport studies were conducted with Cascade Blue (CB, Mw 538), Dextran-Cascade Blue (DCB, Mw 10 kDa), and FITC coupled Dextran (FITC-Dex, Mw 72 kDa). Microneedle arrays with needle lengths of 900, 700 and 550 micro m are able to pierce dermatomed human skin as evident from (a) the appearance of blue spots on the dermal side of the skin after Trypan Blue treatment and (b) elevated TEWL levels after piercing compared to non-treated human dermatomed skin. Microneedles with a length of 300 micro m did not pierce human skin in vitro. Transport studies performed with model compounds ranging from 538 Da to 72 kDa revealed that pretreatment with microneedle arrays enhanced the transport across dermatomed human skin. However, some degradation was also observed for FITC-Dex and DCB. We conclude that assembled microneedle arrays can be used to deliver compounds through the skin up to a molecular weight of at least 72 kDa.

Administration, Cutaneous↗
PubMed1993

Changes in adrenergic nerves and tissue perfusion after freezing injury to the ear skin of rabbits.

The aim of the present study was to investigate by a combined trypan blue and catecholamine fluorescence technique the sequential changes in cutaneous adrenergic nerves, the distribution of blood flow, and vascular permeability at the demarcation line that occurred after experimental cold injury. Nine New Zealand white rabbits weighing 4.0-4.4 kg had frostbite induced by pressing the bottom of a glass bottle 2 cm in diameter filled with liquid nitrogen against the shaved skin of the ear. All the rabbits were anaesthetised with ketamine hydrochloride (Ketamine) and xylazine hydrochloride (Rompun). Specimens were taken one and three days, and two weeks after frostbite. Control samples were taken from the opposite normal ear. Trypan blue was injected into a saphenous vein just before the specimens were taken. The specimens were always taken in the same way and selected so that the probable demarcation line of the frostbite ran across the middle. The glyoxylic acid-induced fluorescence method was used to show the adrenergic nerves. There were no adrenergic nerves around the vessels and no arteriovenous anastomoses in the central area of the injury after one day, but catecholamines had started to accumulate in the adrenergic nerve endings at the margins of the injured area. This accumulation was still more obvious three days after frostbite. Some fluorescent regenerating adrenergic nerves could already be seen at the probable demarcation line two weeks after frostbite. There was increased trypan blue fluorescence near the margin of the injured area after one day, indicating extravasation and vascular damage.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Studies on the mechanism of natural killer cell-mediated cytotoxicity. VII. functional comparison of human natural killer cytotoxic factors with recombinant lymphotoxin and tumor necrosis factor.

The present study was undertaken to evaluate the possible contribution of other cytokines to the lytic activity of NKCF-containing supernatants. We compared some of the functional properties of human NKCF and purified recombinant human rLT and rTNF. It was found that the target cell specificity of rLT was quite different from NKCF in that rLT was neither species specific nor NK specific. Furthermore, antibodies against rLT did not affect the lytic activity of NKCF. These results demonstrate that LT does not significantly contribute to the lytic activity mediated by NKCF. The target specificity of rTNF was found to be related to that of NKCF with the exception of one NK-resistant cell line that was lysed by rTNF in a 20-hr 51Cr-release assay. However, rTNF was not toxic to any of the target cells tested as assessed by trypan blue exclusion in a 20-hr assay unless the targets were labeled with 51Cr. In contrast, NKCF did kill target cells as detected by trypan blue exclusion that were not labeled with 51Cr. Further analysis of this mechanistic difference in the lytic activity of rTNF and NKCF revealed that rTNF in combination with either cycloheximide or mitomycin C but not IFN-gamma could lyse unlabeled U937 target cells. In addition, pretreatment of U937 target cells with nonradioactive Na2CrO4 at concentrations equivalent to that used to 51Cr-labeled cells resulted in their susceptibility to lysis by rTNF as assessed by trypan blue exclusion. These findings suggest that lysis of several susceptible target cells in 20 hr by rTNF requires the presence of additional agents that may be sublethally toxic and/or inhibitory to macromolecular synthesis. Antibody inhibition studies revealed that anti-TNF mediated from partial to complete inhibition of lysis of U937 by unfractionated supernatants containing NKCF. However, fractionation of such supernatants on chromatofocusing columns yielded two distinct peaks of activity eluting in the pH range of 5 to 6 and 7 to 8. Anti-TNF could inhibit the acidic form of NKCF but not the neutral form. It is concluded that NKCF activity is mediated in part by TNF or an antigenically related molecule as well as some other distinct factor(s). The lack of consistent inhibition of NK CMC by anti-TNF suggests that TNF alone is not sufficient to mediate NK activity, or else it is inaccessible to the added antibody.

Antibodies, Monoclonal↗
PubMed1996

Antitumor activity of thevetoside in vitro.

OBJECTIVE: To study the effects of thevetoside (TS), a cardiac glycoside, and an inhibitor of Na+, K(+)-ATPase, on tumor cells cultured in vitro. METHODS: The cytotoxic effects of TS on tumor cells were determined by trypan blue dye exclusion, neutral red vital staining and clonogenic assay. The time-effect relationship and growth inhibition of tumor cells by TS were assayed with trypan blue exclusion method. RESULTS: TS at low doses (0.005-0.1 mg.L-1), with dose dependence, was able to kill SMMC-7721, SGC-7901 and HeLa cells. IC50 values for SMMC-7721, SGC-7901 and HeLa cells were 0.007, 0.011 and 0.018 mg.L-1 by trypan blue dye exclusion test and 0.016, 0.055 and 0.078 mg.L-1 by neutral red vital staining test. TS inhibited the clonal forming rate of SMMC-7721 and SGC-7901 significantly with IC50 values of 0.021 and 0.036 mg.L-1, respectively. Only when the cells were continuously treated with TS for more than 8 hours, the drug-induced cell lethality could be displayed and strengthened quickly. The growth of tumor cells was notably inhibited after they were exposed to 0.1 microgram/ml of TS for 12 hours. All the experimental results of antitumor activity in vitro showed that SMMC-7721 was most sensitive to TS among the three kinds of tumor cells. CONCLUSIONS: TS has cytotoxic action on tumor cells cultured in vitro and this lethal effect must have an action process, in which tumor cells are not dead but suffer from deadly injury and lost the capability of unlimited proliferation.

Adenocarcinoma↗
PubMed2002

Mechanisms of shock wave induced endothelial cell injury.

BACKGROUND AND OBJECTIVES: Medical procedures, for example, laser angioplasty and extracorporeal lithotripsy as well as high-energy trauma expose human tissues to shock waves (SWs) that may cause tissue injury. The mechanisms for this injury, often affecting blood vessel walls, are poorly understood. Here we sought to assess the role of two suggested factors, viz., cavitation or reactive oxygen species (ROS). STUDY DESIGN/MATERIALS AND METHODS: A laser driven flyer-plate model was used to expose human umbilical cord vein endothelial cell (HUVEC) monolayers to SWs or to SWs plus cavitation (SWC). Cell injury was quantified with morphometry, trypan blue staining, and release of (51)Cr from labeled HUVECs. RESULTS: HUVECs, exposed to SWs only, could not be distinguished from controls in morphological appearance or ability to exclude trypan blue. Yet, release of (51)Cr, indicated a significant cell injury (P < 0.05). HUVEC cultures exposed to SWC, exhibited cell detachment and cell membrane damage detectable with trypan blue. Release of (51)Cr was fourfold compared to SW samples (P < 0.01). Signs of cell injury were evident at 15 minutes and did not change over the next 4 hours. No protective effects of ROS scavengers were demonstrated. CONCLUSIONS: Independent of ROS, SWC generated an immediate cell injury, which can explain, for example, vessel wall perturbation described in relation to SW treatments and trauma.

Endothelium, Vascular↗