Polypropylene Microcentrifuge Tubes Research Articles

Investigation of the Effects of Labware Contamination on Mass Spectrometry-Based Human Serum Lipidome Analysis.

Polypropylene microcentrifuge tubes (MCTs) are increasingly used in lipidome sample preparation. In the absence of a comprehensive study evaluating ramifications of plasticware utilization in mass spectrometry-based lipidomic analyses, we conducted a systematic analysis to elucidate potential negative effects ascribable to labware contamination in serum lipidomics. During serum lipid extractions, tested glassware introduced 24 labware contaminants. In contrast, Eppendorf polypropylene MCTs contributed 485 contaminant features, many of which could be erroneously putatively identified as lipids via their m/z values. Eppendorf MCTs contamination engendered severe ion-suppression of 40 low abundance serum lipids, while generating mild to modest lipid ion-suppression across a multitude of higher abundance coeluting lipids. Less compatible polypropylene MCTs from an alternative manufacturer introduced a staggering 2,949 contaminant m/z values, severely affecting 75 coeluting serum lipids and causing more frequent and pronounced ion-suppression instances. Furthermore, by performing serum extractions with varied initial volumes, it was ascertained that labware-induced lipid ion-suppression is a dynamic phenomenon, contingent on both lipid and labware contaminant concentrations where low-abundance lipids are disproportionately impacted by coelutes of suppressive contaminants. In addition to lipid ion-suppression, the identification and quantification of 7 fatty acid endogenous serum lipids were compromised by the leaching of structurally identical surfactants from MCTs. MCTs artificially introduced 10 additional primary amides extraneous to serum samples. Utmost caution is imperative in interpreting data concerning primary amides and fatty acids when employing plastic labware. Through this investigation, we aspire to elevate awareness regarding the pernicious impact of labware contamination on lipidome analysis.

A Practical Method for EPR Dosimetry Using Alanine Powder.

This work investigates alanine powder, an inexpensive and versatile material compared to alanine pellets, as a standardized dosimeter for the alanine-EPR system using a Bruker EMX-Micro spectrometer. The feasibility of this method was investigated, and a calibration curve was produced using 40 dosimeters, which were prepared by tightly packing DL-alanine powder in polypropylene microcentrifuge tubes. The dosimeters were irradiated to doses ranging from 0.2-20 Gy using a 60 Co source. A dosimeter handling and measurement protocol was established for all dosimeters. The dosimetric signal was evaluated by measuring the peak-to-peak height of the central resonance peak, and the dose response of alanine powder dosimeters showed a linear behavior in the investigated dose range with relative errors below 13%. Measurement repeatability and reproducibility were tested to show the errors associated with sample placement in the cavity and with the overall measurement method, with both tests showing relative errors below 7%. As an inexpensive material compared to pellet dosimeters, alanine powder has a strong potential to be used as a standardized material for radiation dosimetry applications. The scope of this work is to present an effective and comprehensive methodology with accompanying analysis scripts for dosimetry with alanine powder that is useful in a wide range of applications and dose requirements.

Instability of corticotropin during long-term storage- myth or reality?

Corticotropin is notorious for its instability. Whereas several studies have investigated its short-term stability in plasma following venous blood sampling, studies on long-term stability are lacking. Here we investigated the long-term storage stability of corticotropin in ethylenediaminetetraacetic acid containing plasma. Specimens from healthy volunteers (neat, spiked) were stored in polypropylene microcentrifuge tubes with socket screw-caps at-20°C and-70°C for up to one and a half years. Corticotropin in plasma was measured using an Abbott research only immunoassay. Separately, specimens from patients were collected during diagnostic routine testing and stored in polystyrene tubes with push-caps at-20°C for up to 6 years. In these samples corticotropin hormone was measured using the Diasorin corticotropin immunoassay. Storage of specimens at-20°C or-70°C for up to one and a half years showed minimal changes (<11%) in corticotropin levels, while storage of patient samples at-20°C for up to 6 years showed a significant (54%) reduction in corticotropin levels. Corticotropin levels are stable in plasma when stored at-20°C for one and a half years using the Abbott research only assay, but with longer storage time a significant reduction in corticotropin levels can be expected. Once specimens are stored for future corticotropin measurements, one should consider storage time, storage temperature and assay differences.

Progranulin Adsorbs to Polypropylene Tubes and Disrupts Functional Assays: Implications for Research, Biomarker Studies, and Therapeutics.

Progranulin (PGRN) is a tightly regulated, secreted glycoprotein involved in a wide range of biological processes that is of tremendous interest to the scientific community due to its involvement in neoplastic, neurodevelopmental, and neurodegenerative diseases. In particular, progranulin haploinsufficiency leads to frontotemporal dementia. While performing experiments with a HIS-tagged recombinant human (rh) PGRN protein, we observed a measurable depletion of protein from solution due to its adsorption onto polypropylene (PPE) microcentrifuge tubes. In this study, we have quantified the extent of rhPGRN adsorption to PPE tubes while varying experimental conditions, including incubation time and temperature. We found that ∼25–35% of rhPGRN becomes adsorbed to the surface of PPE tubes even after a short incubation period. We then directly showed the deleterious impact of PGRN adsorption in functional assays and have recommended alternative labware to minimize these effects. Although the risk of adsorption of some purified proteins and peptides to polymer plastics has been characterized previously, this is the first report of rhPGRN adsorption. Moreover, since PGRN is currently being studied and utilized in both basic science laboratories to perform in vitro studies and translational laboratories to survey PGRN as a quantitative dementia biomarker and potential replacement therapy, the reported observations here are broadly impactful and will likely significantly affect the design and interpretation of future experiments centered on progranulin biology.

The pharmacokinetics of the Fab fragment of a humanized anti‐cocaine monoclonal antibody and its effects on cocaine’s distribution in mice

There is currently no FDA approved treatment for cocaine abuse. Our laboratory has created a humanized anti‐cocaine monoclonal antibody (mAb), h2E2, that has high affinity and specificity for cocaine and is cleared slowly in rodents. The mAb h2E2 sequesters cocaine in the plasma and prevents cocaine entry into the brain in rodents. The antigen binding portion, the Fab fragment, of h2E2 has the same high affinity and selectivity as its whole mAb counterpart. The Fab fragment should also sequester cocaine into the plasma compartment but is expected to have a more rapid clearance than the intact mAb. This study elucidated the pharmacokinetics of the Fab fragment and its effects on cocaine’s pharmacokinetics in mice. Catheterized mice were injected with Fab (80 mg/kg, i.v., pH=7.4, PBS) (n=8) and blood samples collected from the tip of the tail at designated time points over three days. Fab concentrations in blood samples were quantified using an ELISA assay. Two groups of catheterized mice were injected under mild restraint with Fab (82 mg/kg, i.v. pH= 7.4, PBS, generated by enzymatic digestion of whole mAb h2E2) or vehicle (PBS, pH=7.4). One hour later, mice were injected with an equimolar dose of cocaine HCl (0.56 mg/kg, i.v.). At 8 time points between 0.75 and 60 min, mice were decapitated, and trunk blood was collected. Sodium pentobarbital (50 mg/kg, i.p.) was administered 3 minutes prior to decapitation. For the 0.75 and 1.5‐minute time points, cocaine was injected into anesthetized rats. Blood (typically 0.5–2 mL) was placed into 2‐mL polypropylene tubes and then centrifuged at 5000 g for 3 minutes. Plasma was pipetted into polypropylene microcentrifuge tubes and rapidly frozen on dry ice and stored at −80°C until LC‐MS/MS analysis. This experiment was performed three times. Total cocaine concentrations were quantified using LC‐MS/MS. The pharmacokinetic profile of the Fab fragment in mice was described by a 2‐compartment model (Figure 1). The Fab fragment has a mean distribution half‐life (t1/2α) of 16.7 minutes and a terminal elimination half‐life (t1/2β) of 7.1 hours (Figure 1). In the presence of the Fab fragment, cocaine concentrations in the plasma increased by 4.5‐fold compared to vehicle animals (Figure 2). The initial volume of distribution of cocaine decreased approximately 4.5‐fold in the presence of Fab. The Fab fragment increases the concentration of cocaine in the plasma by approximately 4.5‐fold compared to vehicle controls. Although the data is not shown, it is hypothesized that the Fab fragment, like its mAb counterpart, prevents cocaine from crossing the blood‐brain barrier and entering the brain. With a 24‐fold decrease in elimination half‐life (7.1 hours) compared to the intact h2E2 (7.8 days) and predicted rapid urinary excretion, the Fab fragment may be useful for the treatment of cocaine overdoses.Support or Funding InformationThis work is supported by the National Institutes of Health, National Institute on Drug Abuse grant number U01DA039550 (to ABN).The pharmacokinetics of the Fab fragment in male miceFigure 1The pharmacokinetics of cocaine in the presence of the Fab fragmentFigure 2

Storing of Extracts in Polypropylene Microcentrifuge Tubes Yields Contaminant Peak During Ultra-flow Liquid Chromatographic Analysis.

Background and Aim:This study was designed to understand the effect of storage in polypropylene microcentrifuge tubes and glass vials during ultra-flow liquid chromatographic (UFLC) analysis.Materials and Methods:One ml of methanol was placed in polypropylene microcentrifuge tubes (PP material, Autoclavable) and glass vials (Borosilicate) separately for 1, 2, 4, 8, 10, 20, 40, and 80 days intervals stored at −4°C.Results:Contaminant peak was detected in methanol stored in polypropylene microcentrifuge tubes using UFLC analysis. The contaminant peak detected was prominent, sharp detectable at 9.176 ± 0.138 min on a Waters 250–4.6 mm, 4 μ, Nova-Pak C18 column with mobile phase consisting of methanol:water (70:30).Conclusion:It was evident from the study that long-term storage of biological samples prepared using methanol in polypropylene microcentrifuge tubes produce contaminant peak. Further, this may mislead in future reporting an unnatural compound by researchers.SUMMARY Long-term storage of biological samples prepared using methanol in polypropylene microcentrifuge tubes produce contaminant peakContamination peak with higher area under the curve (609993) was obtained in ultra-flow liquid chromatographic run for methanol stored in PP microcentrifuge tubesContamination peak was detected at retention time 9.113 min with a lambda max of 220.38 nm and 300 mAU intensity on the given chromatographic conditionsGlass vials serve better option over PP microcentrifuge tubes for storing biological samples. Abbreviations used: UFLC: Ultra Flow Liquid Chromatography; LC: Liquid Chromatography; MS: Mass spectrometry; AUC: Area Under Curve.

A simple and inexpensive device for biofilm analysis

The Calgary Biofilm Device (CBD) has been described as a technology for the rapid and reproducible assay of biofilm susceptibilities to antibiotics. In this study a simple and inexpensive alternative to the CBD was developed from polypropylene (PP) microcentrifuge tubes and pipette tip boxes. The utility of the device was demonstrated using Candida glabrata, a yeast that can develop antimicrobial-resistant biofilm communities. Biofilms of C. glabrata were formed on the outside surface of microcentrifuge tubes and examined by quantitative analysis and scanning electron microscopy. Growth of three C. glabrata strains, including a clinical isolate, demonstrated that biofilms could be formed on the microcentrifuge tubes. After 24h incubation the three C. glabrata strains produced biofilms that were recovered into cell suspension and quantified. The method was found to produce uniform and reproducible results with no significant differences between biofilms formed on PP tubes incubated in various compartments of the device. In addition, the difference between maximum and minimum counts for each strain was comparable to those which have been reported for the CBD device.

Adsorption and Degradation of Doxorubicin from Aqueous Solution in Polypropylene Containers

The purpose of this study was to examine doxorubicin adsorption in polypropylene containers as a function of pH and drug concentration based on anecdotal evidence of such adsorption. Doxorubicin loss was first examined in high-performance liquid chromatography (HPLC) glass inserts by UV absorbance to determine appropriate pH and time durations for subsequent analysis. Doxorubicin loss was then investigated in polypropylene microcentrifuge tubes at different pH values and starting drug concentration at 37°C over 48h using HPLC with fluorescent detection. Doxorubicin concentrations was essentially constant in HPLC glass inserts at pH 4.8 up to 12h but declined 5% at pH 7.4 by 3h. The percent doxorubicin adsorption was calculated in polypropylene microcentrifuge tubes from extrapolations to zero time and was the least at pH 4.8, but increased with pH values 6.5 and 7.4, and decreased with drug concentration to reach a maximum adsorption of 45% in 2.0μg/mL at pH 7.4 and 37°C. Degradation rate constants, ranging from 0.0021 to 0.019h(-1), also increased with pH in these studies. Determinations of low amounts of doxorubicin in polypropylene containers at pH 7.4 may be underestimated if adsorption and degradation issues are not taken into account.

Oral Symptom Intensity, Health-Related Quality of Life, and Correlative Salivary Cytokines in Adult Survivors of Hematopoietic Stem Cell Transplantation with Oral Chronic Graft-versus-Host Disease

Oral chronic graft-versus-host disease (cGVHD) is a frequent, clinically significant sequela of allogeneic hematopoietic stem cell transplantation (HSCT). This study was designed to elucidate relationships among clinical characteristics of oral cGVHD and related oral pain and oral dryness, salivary proinflammatory cytokine interleukin (IL)-6 and IL-1alpha concentrations, and health-related quality of life (HRQL). An understanding of the characteristics and correlates of oral cGVHD manifestations and related symptoms, such as oral dryness, is fundamental to the development of therapeutic interventions. Oral cGVHD severity was assessed with the Oral Mucositis Rating Scale (OMRS). Oral pain and perceived intensity of oral dryness were self-reported via a visual analog scale and a numeric rating scale, respectively. HRQL was assessed with the Functional Assessment of Cancer Therapy-General (FACT-G). Salivary IL-1alpha and IL-6 concentrations were measured by enzyme-linked immunosorbent assay. All 42 adult subjects (59% males) had clinician-assessed oral cGVHD by the OMRS scale (mean score, 18.38 +/- 12.99; range, 2-46). Oral dryness (in 43% of subjects; mean OMRS score, 2.56 +/- 3.45; range, 0-10) was more prevalent than oral pain (8%; mean score, 0.13 +/- 0.47). Salivary IL-6 was associated with oral cGVHD severity (r = 0.49; P < .01), oral ulceration (r = 0.38; P = .04), and erythema (r = 0.63; P < .01). FACT-G total score and physical and emotional well-being subscale scores were meaningfully lower than U.S. population normative values. Participants with more severe oral cGVHD manifestations had significantly inferior social/family well being (r = -0.49; P < .01). Oral dryness was associated with higher salivary IL-1alpha (r = 0.41; P = .04) and, controlling for cGVHD severity, with lower HRQL (r = -0.41; P = .03). Subjects with moderate to severe oral dryness tended to report the poorest overall HRQL. This study provides preliminary evidence of the relationship between oral dryness and HRQL, the contribution of oral cGVHD to inferior HRQL, and the association between IL-6 and oral cGVHD severity, ulceration, and erythema. The high prevalence of oral dryness and its relationship to HRQL in a sample of subjects with oral cGVHD underscores the importance of improving our evaluation and management of this symptom in long-term survivors of allogeneic HSCT. The positive associations between IL-6 and oral cGVHD severity and erythema, as well as the positive trend with oral ulceration, warrant further exploration of this cytokine as a potential biomarker of active oral cGVHD.

The Fuge Tube Diode Array Spectrophotometer

We present the details for adapting a diode array UV�vis spectrophotometer to incorporate the use of polypropylene microcentrifuge tubes�fuge tubes�as cuvettes. Optical data are presented validating that the polyethylene fuge tubes are equivalent to the standard square cross section polystyrene or glass cuvettes generally used in spectrophotometers. The fuge tubes also can serve as reaction vessels, which permits the ready study of chemical systems involving heterogeneous phases by eliminating the need for tedious separations.

Identification of Binding Sites in the Nicotinic Acetylcholine Receptor for TDBzl-etomidate, a Photoreactive Positive Allosteric Effector

Etomidate, one of the most potent general anesthetics used clinically, acts at micromolar concentrations as an anesthetic and positive allosteric modulator of gamma-aminobutyric acid responses, whereas it inhibits muscle-type nicotinic acetylcholine receptors (nAChRs) at concentrations above 10 microm. We report here that TDBzl-etomidate, a photoreactive etomidate analog, acts as a positive allosteric nAChR modulator rather than an inhibitor, and we identify its binding sites by photoaffinity labeling. TDBzl-etomidate (>10 microm) increased the submaximal response to acetylcholine (10 microm) with a 2.5-fold increase at 60 microm. At higher concentrations, it inhibited the binding of the noncompetitive antagonists [(3)H]tetracaine and [(3)H]phencyclidine to Torpedo nAChR-rich membranes (IC(50) values of 0. 8 mm). nAChR-rich membranes were photolabeled with [(3)H]TDBzl-etomidate, and labeled amino acids were identified by Edman degradation. For nAChRs photolabeled in the absence of agonist (resting state), there was tetracaine-inhibitable photolabeling of amino acids in the ion channel at positions M2-9 (deltaLeu-265) and M2-13 (alphaVal-255 and deltaVal-269), whereas labeling of alphaM2-10 (alphaSer-252) was not inhibited by tetracaine but was enhanced 10-fold by proadifen or phencyclidine. In addition, there was labeling in gammaM3 (gammaMet-299), a residue that contributes to the same pocket in the nAChR structure as alphaM2-10. The pharmacological specificity of labeling of residues, together with their locations in the nAChR structure, indicate that TDBzl-etomidate binds at two distinct sites: one within the lumen of the ion channel (labeling of M2-9 and -13), an inhibitory site, and another at the interface between the alpha and gamma subunits (labeling of alphaM2-10 and gammaMet-299) likely to be a site for positive allosteric modulation.

Post‐collection,pre‐measurement variables affecting VEGF levels in urine biospecimens

Angiogenesis, the development and recruitment of new blood vessels, plays an important role in tumour growth and metastasis.Vascular endothelial growth factor (VEGF) is an important stimulator of angiogenesis.Circulating and urinary VEGF levels have been suggested as clinically useful predictors of tumour behaviour, and investigations into these associations are ongoing.Despite recent interest in measuring VEGF levels in patients, little is known about the factors that influence VEGF levels in biospecimens. To begin to address this question, urine samples were collected from patients with solid tumours undergoing radiotherapy and healthy volunteers.Four factors were examined for their effects on VEGF concentrations as measured by chemiluminescent immunoassay: time from sample collection to freezing, number of specimen freeze–thaw cycles, specimen storage tube type and the inclusion or exclusion of urinary sediment. The results of this study indicate that time to freeze up to 4 hrs, number of freeze–thaw cycles between one and five, and different types of polypropylene tubes did not have statistically significant effects on measured urinary VEGF levels. Urinary sediment had higher VEGF levels than supernatant in five of six samples from healthy patients.It is not clear whether there is an active agent in the sediment causing this increase or if the sediment particles themselves are affecting the accuracy of the assay.Therefore, we recommend centrifuging urine, isolating the supernatant, and freezing the sample in polypropylene microcentrifuge tubes or cryogenic vials within 4 hrs of collection.In addition, we recommend the use of samples within five freeze–thaw cycles.

Distinctive T Cell-suppressive Signals from Nuclearized Type 1 Sphingosine 1-Phosphate G Protein-coupled Receptors

Sphingosine 1-phosphate (S1P) generated by cells of innate immunity and the type 1 S1P G protein-coupled receptor (S1P(1)) on mobile T cells constitute a major system for control of lymphoid organ traffic and tissue migration of T cells. Now we show that T cell activation mediated by the T cell antigen receptor translocates plasma membrane S1P(1) to nuclear envelope membranes for association there with G(i/o), Erk (1/2), and other proteins that plasma membrane S1P(1) uses to signal T cell proliferation. However, nuclear S1P(1) and plasma membrane S1P(1) transduce opposite effects of S1P on T cell proliferation and relevant signaling as exemplified by respective decreases and increases in T cell nuclear concentrations of both phospho-Erk and active (phosphorylated) c-Jun. T cell antigen receptor-mediated activation of T cells therefore both eliminates migration responses to S1P by down-regulation of plasma membrane S1P(1) and translocates the S1P-S1P(1) axis into the nuclear domain where signals are directed to transcriptional control of immune functions other than migration.

Determination of the Substrate Specificity of Tripeptidyl-peptidase I Using Combinatorial Peptide Libraries and Development of Improved Fluorogenic Substrates

Classical late-infantile neuronal ceroid lipofuscinosis is a fatal neurodegenerative disease caused by mutations in CLN2, the gene encoding the lysosomal protease tripeptidyl-peptidase I (TPP I). The natural substrates for TPP I and the pathophysiological processes associated with lysosomal storage and disease progression are not well understood. Detailed characterization of TPP I substrate specificity should provide insights into these issues and also aid in the development of improved clinical and biochemical assays. To this end, we constructed fluorogenic and standard combinatorial peptide libraries and analyzed them using fluorescence and mass spectrometry-based activity assays. The fluorogenic group 7-amino-4-carbamoylmethylcoumarin was incorporated into a series of 7-amino-4-carbamoylmethylcoumarin tripeptide libraries using a design strategy that allowed systematic evaluation of the P1, P2, and P3 positions. TPP I digestion of these substrates liberates the fluorescence group and results in a large increase in fluorescence that can be used to calculate kinetic parameters and to derive the substrate specificity constant kcat/KM. In addition, we implemented a mass spectrometry-based assay to measure the hydrolysis of individual peptides in peptide pools and thus expand the scope of the analysis. Nonfluorogenic tetrapeptide and pentapeptide libraries were synthesized and analyzed to evaluate P1' and P2' residues. Together, this analysis allowed us to predict the relative specificity of TPP I toward a wide range of potential biological substrates. In addition, we evaluated a variety of new fluorogenic peptides with a P3 Arg residue, and we demonstrated their superiority compared with the widely used substrate Ala-Ala-Phe-AMC for selectively measuring TPP I activity in biological specimens.

Impurities from polypropylene microcentrifuge tubes as a potential source of interference in simultaneous analysis of multiple lipid-soluble antioxidants by HPLC with electrochemical detection.

High-performance liquid chromatography (HPLC) with electrochemical detection was used to analyze lipid-soluble antioxidants and micronutrients in plasma. Small amounts of plasma samples are often extracted in polypropylene (PP) microcentrifuge tubes before HPLC analysis due to its convenience. We therefore investigated the effect of impurities released from different PP tubes during extraction on the electrochemical detection of retinol, lutein, alpha-tocopherol, gamma-tocopherol, retinyl palmitate, beta-carotene and total coenzyme Q10 by an HPLC system with a coulometric array detector (CoulArray HPLC). An array of eight electrical potentials and gradient elution were applied. Human plasma or ultrapure water were extracted in four different PP tubes. Peaks at different retention times with varied amounts of unknown compounds were detected from different PP tubes. Although all those unknown peaks could be differentiated from peaks of interest by the CoulArray HPLC, they caused busy chromatographs, and one peak had an overlap with the dominant peak of lutein. Moreover, that peak completely masked the lutein peak when only one oxidizing potential was applied. Our results suggest potential interference from PP tubes on electrochemical detection especially when using only one electrical potential or having poor separation for these antioxidants if PP tubes are not carefully tested.

High-Efficiency Solid-Phase Capture Using Glass Beads Bonded to Microcentrifuge Tubes: Immunoprecipitation of Proteins from Cell Extracts and Assessment of Ras Activation

We have bonded glass microbeads (425–600 μm diameter) to the inner walls of polypropylene microcentrifuge tubes. In addition to increasing the surface area of the tubes manyfold, the beads provide surface Si groups which can be reacted with a silane compound such as aminopropyltriethoxysilane, yielding a free amino group. The amino group is reacted with another cross-linking reagent, for example, the homobifunctional compound dimethyl suberimidate, which can form a covalent bond with amine groups of proteins. After binding protein A or G to the dimethyl suberimidate, the beads were used to immunoprecipitate proteins from cell extracts; we show that the protein A/G-coated glass beads yield similar amounts of immunoprecipitated proteins as a standard method using protein A– or G–agarose beads, but with fewer contaminating proteins. In addition, we show that when immunoprecipitating Ras from cell extracts and measuring the amounts of Ras-bound GTP and GDP, the new method yielded higher guanine nucleotide levels than protein G–agarose beads, suggesting that it caused less denaturation of Ras. Because the glass beads are bonded to the walls of the tubes, the immunoprecipitates can be washed rapidly and efficiently, and we show that 20–30 tubes can be washed in 1/10 the time required to wash immunoprecipitates on protein A– or G–agarose beads.

Getting Suspended Cells or Particles Embedded in the Tip of BEEM Capsules

Abstract It can be a problem to get suspended (or plated) cells or particles to settle into the apex of a BEEM capsule for embedding, particularly if the cei is/particles can not be embedding in agar or gelatin for some reason. The following simple procedure solves this problem. Process the suspensions in polypropylene test tubes or microcentrifugation tubes. These can be spun down after each transfer for 5 minutes at 3000 rpm. Once the cells/particles are fixed, en bloc stained, and infiltrated with either Spurr's or LR White resin, I transfer them to BEEM capsules. I do not add additional resin yet. These are placed piggyback in microcentrifuge tubes that have been slit with wire cutters to make them large enough for the BEEM capsules to fit (see following illustration). Processing cell suspensions or plated cells can be done in polypropylene test tubes or microcentrifuge tubes. These can be spun down after each transfer for 5 minutes at 3000 rpm. Once the cells are fixed, enbloc stained, and infiltrated with either Spurr or LRWhite resin, I transfer them to BEEM capsules. I do not add additional resin yet.

Undesirable Reaction of Aqueous Cyclodextrin Solutions with Polypropylene

Cyclodextrins are cyclic sugars that have extensive applications in the pharmaceutical, food, biomedical, and other industries. Although cyclodextrins are water-soluble sugars, they have hydrophobic central cavities that are capable of accommodating nonpolar molecules of the right dimensions. This inclusion complexation property, which is key to its utilization in solubilization and stabilization of nonpolar guest molecules, can also lead to undesirable reactions. The present investigation demonstrates the ability of aqueous cyclodextrin solutions to extract plastic additives when stored in polypropylene tubes that are commonly used in biological research. Storage of α- and γ-cyclodextrin solutions in polypropylene microcentrifuge tubes led to the extraction of 2,6-di- tert-butyl- p-cresol, or BHT, a common additive used as an antioxidant and for polymer stabilization. γ-Cyclodextrin solutions also formed an insoluble complex with an unidentified substance. These results suggest the potential of cyclodextrin solutions stored in plastic containers to be contaminated with additives. In many cases, the additives added by plastic manufacturers may be trade secrets and difficult to identify. One should be cautious in using aqueous cyclodextrin solutions stored in plastic containers for biological or biomedical research and applications.

Isolation and culture of highly enriched populations of Leydig cells from guinea-pig (Cavia porcellus) testes.

Leydig cells were isolated from adult male guinea-pig testes using a multi-step procedure involving enzymatic dissociation and Percoll-gradient centrifugation. The following description is the first account of a successful isolation of adolescent guinea-pig Leydig cells. The enriched Leydig-cell preparation routinely isolated from six intact testicles yielded approximately 5.0 x 10(6) +/- 0.7 x 10(6) (+/- SEM) Leydig cells with a viability of 98.0 +/- 0.4% as determined using the trypan-blue exclusion method. The purity of the isolated cell population as assessed by 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) staining averaged 82.5 +/- 0.8%. Under light microscopy, guinea-pig Leydig cells were polyhedral in shape with a large prominent nucleus and a distinct nucleolus. The acidophilic cytoplasm contained numerous lipid-filled vesicles. Ultrastructurally, guinea-pig Leydig cells displayed an eccentrically located ovoid nucleus with dark-staining peripheral heterochromatin. Large quantities of mitochondria, smooth endoplasmic reticulum and particulate-laden lipid droplets were also evident. The steroidogenic potential of the isolated Leydig cells was verified using a maximally stimulating dose of ovine LH (100 ng ml-1) and human CG (200 mIU ml-1). Leydig cells incubated in a shaking (120 cycles min-1) water bath for 3 h at 37 degrees C in capped polypropylene microcentrifuge tubes produced 233 +/- 21 ng and 223 +/- 18 ng testosterone per 1 x 10(6) cells when maximally stimulated with oLH or hCG, respectively. The inclusion of low (1-5 microM) levels of sodium ascorbate during culture enhanced significantly Leydig-cell viability vs. control values.

Hydrophobic Binding of Pertussis Toxin Is Enhanced by Oligosaccharide Receptors

Pertussis toxin is one of several virulence factors produced by Bordetella pertussis, the etiologic agent of whooping cough. Pertussis toxin is an oligomeric A-B class toxin composed of an ADP-ribosyltransferase S1 (A) subunit and a B oligomer containing lectin-like binding domains. The carbohydrate binding specificity of the B oligomer is for sialooligosaccharide sequences expressed on target cell receptors and asparagine-linked glycans found in many serum glycoproteins. Pertussis toxin also has the ability to bind to the inert surfaces of culture tubes. In this report we present data showing that pertussis toxin binding to polypropylene microcentrifuge tubes was enhanced in a time- and concentration-dependent manner by the addition of soluble glycoprotein or oligosaccharide receptor analogs. Evidence obtained using the hydrophilic and hydrophobic surfaces of Gel Bond electrophoresis casting film indicated that receptor-enhanced binding was likely due to hydrophobic interactions. Hydrophobic binding of the isolated B oligomer of pertussis toxin was enhanced only in the presence of high concentrations of glycoproteins. Therefore, the S1 (A) subunit of pertussis holotoxin appears to play a role in receptor-enhanced hydrophobic binding. We propose, therefore, that pertussis toxin binding to its receptors may expose or preferentially orient hydrophobic residues that may contribute to the functional association of the toxin with host cell plasma membranes and delivery of the S1 subunit to its intracellular target.