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D Redelman

Publications and source records attributed to D Redelman.

At least 19 recordsLinked to original sources
PubMed1998

Titration of a CD45-FITC conjugate to determine the linearity and dynamic range of fluorescence intensity measurements on lymphocytes.

To produce biologic calibrators for relative fluorescence intensity (RFI) measurements, we stained leukocytes with serial dilutions of CD45-FITC conjugate and processed them using our regular whole blood lysis procedure. Cells were stained with conjugate concentrations ranging from twice recommended to a million-fold lower. At the highest concentrations of conjugate, the RFI reached a plateau near the top of the third decade, indicating saturation of CD45 binding sites. As the concentration decreased, the RFI declined in a highly linear relationship between the dilution factor and the histogram channel number. For channel numbers corresponding to the lowest percentiles of the RFI distribution, linearity persisted down to the first half decade. The slope of this relationship revealed a true dynamic range of 4.5 decades, which was comparable to the value obtained with microbead standards calibrated in molecules of equivalent soluble fluorochrome (MESF). Our results suggest that the lower limit of linearity for fluorescence intensity from fluorescein isothiocyanate (FITC)-stained lymphocytes is below 500 MESF and that cellular autofluorescence is the major limiting factor in detecting and quantifying FITC-specific staining. This procedure provides an adroit way of characterizing the linearity and dynamic range of measurements for quantitative fluorescence cytometry using exactly the same matrix, stains, and preparation methods as those used for cellular analytes.

Antibodies, Monoclonal↗
PubMed1997

Proposed new data file standard for flow cytometry, version FCS 3.0.

In 1984, the first flow cytometry data file format was proposed as Flow Cytometry Standard 1.0 (FCS1.0). FCS 1.0 provided a uniform file format allowing data acquired on one computer to be correctly read and interpreted on other computers running a variety of operating systems. That standard was modified in 1990 and adopted by the Society of Analytical Cytology as FCS 2.0. Here, we report on an update of the FCS 2.0 standard which we propose to designate FCS 3.0. We have retained the basic four segment structure of earlier versions (HEADER, TEXT, DATA and ANALYSIS) in order to maintain analysis software compatibility, where possible. The changes described in this proposal include a method to collect files larger than 100 megabytes (not possible in earlier versions of the standard), the inclusion of international characters in the TEXT portions of the file, a method of verifying data integrity using a 16-bit cyclic redundancy check, and increased keyword support for cluster analysis and time acquisition. This report summarizes the work of the ISAC Data File Standards Committee. The complete and detailed FCS 3.0 standard is available through the ISAC office [Sherwood Group, 60 Revere Drive, Ste 500, Northbrook, IL 60062, phone: (847) 480-9080 ext. 231, fax: (847) 480-9282, E-mail: isac@sherwood-group.com] or through the internet at the ISAC WWW site, http://nucleus.immunol.washington.edu/ISAC.ht ml.

Database Management Systems↗
PubMed1996

Determination of activable proacrosin/acrosin in bovine sperm using an irreversible isocoumarin serine protease inhibitor.

The activable proacrosin/acrosin levels in bovine sperm were examined using fluorescent staining and flow cytometry. The proportion of sperm with active acrosin were determined using the biotinylated isocoumarin serine protease inhibitor, Bi-Aca-Aca-OMe-IC (BIC). The presence of bound inhibitor on sperm was then determined by secondary labeling with avidin fluorescein conjugate. The proportion of sperm with activable proacrosin/acrosin was assessed by using detergent treatment to expose the active acrosin in intact sperm. The difference between untreated and detergent-treated aliquots was used to estimate the proportion of sperm with activable proacrosin/acrosin. In the 24-h stored samples from six bulls, the mean proportion of sperm with activable proacrosin/acrosin was 78.8 +/- 2.8%, whereas the mean proportion with exposed acrosin after cryopreservation of these samples was 55.8 +/- 4.1%. Significant differences (p < 0.05) were found among bulls in the proportion of sperm with activable proacrosin/acrosin both before and after cryopreservation. Activable proacrosin/acrosin levels in samples of cryopreserved sperm from five bulls were not correlated with fertility. These results do indicate, however, that the irreversible isocoumarin serine protease inhibitor BIC can be used to determine the proportion of sperm cells that retain activable proacrosin/acrosin after cryopreservation and thawing.

Acrosin↗
PubMed1994

Computing the central location of immunofluorescence distributions: logarithmic data transformations are not always appropriate.

The idea of the "average" intensity of immunofluorescence data is often poorly defined, with such terms as average, mean, and peak used interchangeably. In addition, the common use of logarithmic amplifiers with immunofluorescence data further complicates the problem. Log amplifiers permit the display of a wider range of fluorescence intensities. At the same time, they effect a log transformation of the data. This transformation decreases the variance resulting in narrower fluorescence distributions, which are assumed to approximate normal distributions. When the log transform is used, the distribution mean is the geometric mean of the untransformed data, which is computed simply as the mean of the channel values. This mean value serves as a simple indicator of the population center. Despite the prevalence of log transformations in flow cytometry, this transformation may not yield normally distributed immunofluorescence data, whereas the square root or other fractional power transformations can yield normal distributions.

Flow Cytometry↗
PubMed1994

Cell subset (CS) parameter to record the identities of individual cells in flow cytometric data.

The Flow Cytometry Standard (FCS) for cytometric data (Dean et al.: Cytometry 11:323-332, 1990) provides for appending an ANALYSIS section to a data file, but it does not explicitly provide for recording the identities of individual cells. We propose an extension to the FCS definition in order to record the identity of each individual cell in list mode data. In order to do so, one first defines the subpopulations of cells to be identified and one than assigns a number to each defined population. For example, in an analysis in which peripheral blood mononuclear cells are labeled with antibodies to CD3, CD4, and CD8, the "negative" lymphocytes that are labeled with none of the antibodies could be identified as population 1, the CD3-CD4-CD8+ lymphocytes as population 2, etc. As the measured values from each cell are analyzed, the number that identifies the population to which that cell belongs is assigned as the value of an additional parameter. Since this procedure merely adds a new parameter, the only necessary extension to the FCS specification is that a particular name is recognized for this parameter. We propose that CS (abbreviation for cell subset) be recognized as the name and that CS be used as the value for the $PnN (parameter name) keyword in the FCS file TEXT. The CS values that are created can be used to aid in data analysis and can be permanently recorded in a conventional FCS file. A data file that is saved with CS values includes an explicit and integral record of the complete analysis, regardless of the complexity of the analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

CD3 Complex↗
PubMed1992

Development and characterization of a monoclonal antibody specific for the bovine low-affinity interleukin-2 receptor, BoCD25.

An IgM monoclonal antibody, UC-2C2 was produced using splenocytes from mice immunized with cultures of interleukin-2 (IL-2)-dependent bovine peripheral blood lymphocytes. UC-2C2 was found to recognize a cell surface antigen of apparent molecular weight 52,000-54,000 present on activated bovine peripheral blood mononuclear leucocytes (PBML) but not on resting PBML or cells of the bovine lymphoblastoid cell line BL3. The 52,000-54,000 MW antigen was expressed early following activation of PBML by mitogens or alloantigens, with the majority of cells positive by 48 h of culture. UC-2C2 was unable to block binding of phycoerythrin (PE)-conjugated human recombinant IL-2 to PHA-stimulated bovine PBML as determined by flow cytometric analysis. However, two-colour analyses indicated that the antigen recognized by UC-2C2 was present on the same cell population that expressed IL-2 receptors. All activated T lymphocytes of BoCD4, BoCD8 and gamma delta receptor positive phenotypes expressed the target antigen of UC-2C2 and IL-2 receptors. Monocytes and B lymphocytes expressed the target antigen of UC-2C2 and IL-2 receptors at a lower density. This differential expression by the various PBML subpopulations parallels that described for expression of the low-affinity IL-2 receptor (CD25) on human leucocyte subpopulations. Based upon the relative molecular weight, time-course of expression and cellular distribution of the antigen identified by UC-2C2, it is inferred that UC-2C2 recognizes an epitope on the bovine homologue of CD25 which is not involved in binding IL-2.

Animals↗
PubMed1991

Metabolic toxicity of fluorescent stains on thawed cryopreserved bovine sperm cells.

Several fluorescent probes, including derivatives of carboxyfluorescein, carbocyanine, ethidium, and rhodamine, have been used to assess sperm viability. However, the effects of these fluorescent dyes on the metabolic activity of sperm cells have not been systematically examined. This study was conducted to determine the effect of specific fluorescent stains on the metabolic processes of sperm. Cryopreserved bovine sperm cells were thawed, fluorescently stained, and examined using metabolic and flow cytometric techniques. Sperm were stained with either rhodamine 123 (Rhod-123), the aliphatic cell-tracking compound PKH2-GL, dihydro-ethidium (HED), the bisbenzimide stain Hoechst 33342 (Ho33342), or left unstained. The stained samples were compared for metabolic activity, cell staining pattern, and fluorescent intensity over a 180-min period. Samples stained with HED, Ho33342, and PKH2-GL had less oxygen uptake when compared with the unstained sperm samples (p greater than 0.05). Unstained samples and samples stained with Rhod-123 had similar oxygen consumption. The carbon dioxide produced during the 180 min was not different between controls and stained samples. Therefore, some fluorescent probes inhibit the oxygen metabolism of thawed, cryopreserved bovine sperm cells.

Animals↗
PubMed1991

The effect of cottonseed components on testicular development in pubescent rams.

The effect of gossypol on testicular development in rams was examined using 2 experimental approaches. Trial I utilized direct ruminal deposition of gossypol acetic acid (GAA; 95% purity), whereas Trial II compared 2 types of dietary cottonseed, regular and glandless. For Trial I, 18 Polypay or Polypay x Dorset ram lambs, aged 4 to 6 mo, were surgically fitted with rumen cannulae. These 18 ram lambs were divided into groups of 6 which were given either 0, 2.2 or 6.6 mg of GAA/kg body weight daily via gelatin capsules directly into the rumen. All rams in Trial I were slaughtered after 4 or 8 w of treatment to allow recovery of their scrotal contents. The mean testicular weight for the group of rams given 6.6 mg GAA/kg/d was 8% (p less than .05) less than for the rams given 0 mg GAA/kg/d. The caudal epididymal sperm were examined microscopically for percentage of progressively motile sperm and flow cytometrically for mitochondrial function using rhodamine 123 (R123). Differences in sperm motility were not evident among the treatment groups, nor were differences detectable in the 2 major sperm populations, B and C, that were identified by flow cytometry. In Trial II, 21 Polypay and Polypay-Dorset ram lambs, aged 10 mo, were randomly assigned into 3 groups (n = 7) and fed either glandless fuzzy cottonseed (GFC), glandless flake cottonseed (GFL) or regular fuzzy cottonseed (RFC). All rams were fed for 5 w a total daily ration (TDR) equivalent to 3.5% of their body weight daily with 25% being cottonseed. After 8 w the scrotal contents were recovered by castration.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Assessment of the viability and fertilizing potential of cryopreserved bovine spermatozoa using dual fluorescent staining and two-flow cytometric systems.

A dual fluorescent staining system utilizing 5 (and-6)-carboxy-4',5'-dimethyl fluorescein diacetate (CDMFDA) and Hydroethidine (HED) was developed to provide quantifiable information reflective of spermatozoal viability and fertilizing potential. Cryopreserved spermatozoa from ten bulls on which there was fertilizing capacity information were incubated for 1.5, and 3 hr at 39 degrees C prior to fluorogenic staining. Spermatozoa were analyzed using both a FACS Analyzer and an EPICS V flow cytometer to determine if a particular fluorescence pattern was due to an instrumental artifact or cellular processes. Five fluorescent cellular populations were identified by the FACS Analyzer and three populations by the EPICS V. Spermatozoa were quantified after each incubation time for red (HED) and green (CDMFDA) fluorescence. Viable spermatozoa retained the greatest amount of both green and red fluorescence. Dead or moribund spermatozoa had a decrease in over-all fluorescence. The number of viable cells at 0 hr plus the number of dead or morbid cells at any time period were identified by the FACS Analyzer as important in estimating the potential fertility of a bull. The EPICS V identified the number of dead or moribund cells as being related to nonreturn rates. Incubation of samples decreased cellular viability, which resulted in reduced levels of both green and red fluorescence. Similarities between data obtained with both flow cytometers illustrated that cellular processes, not instrumental artifacts, were responsible for the decrease in over-all fluorescence when viability declined, the relationship between the number of cells with specific fluorescence levels and nonreturn rates, and the incubative-induced changes in fluorescence patterns.

Animals↗
PubMed1989

Experimental vesicular stomatitis virus infection of swine: extent of infection and immunological response.

Swine, a natural host species for infection by vesicular stomatitis virus (VSV), were infected with VSV-New Jersey (VSV-NJ) serotype virus obtained from a recent field isolate. Tissues collected from the infected pigs were examined for the presence of infective virus, for viral antigens, and/or for viral nucleic acid. Infective virus could be recovered from tissues near the site of infection for as long as 6 days after the primary infection with VSV. However, no infective virus was recovered following hypothermia induced 11 weeks after infection, or following a secondary challenge with virus 22 weeks after initial infection. Immunofluorescence tests for viral antigens and nucleic acid hybridization assays failed to detect viral antigens or nucleic acids in tissues from which no infective virus could be recovered. Titers of serum-neutralizing antibody peaked 3-5 weeks after infection and then fell slightly until the secondary infection which caused a rapid anamnestic response. Peripheral blood mononuclear cells (PBM) tested 3, 5, 8 or 18 weeks after primary infection all produced readily detectable antigen-specific proliferative responses when cultured with VSV. Thus, although direct tests failed to demonstrate persistence of virus after infection, the humoral and cellular immune response remained elevated for months. Infective VSV was not required to stimulate the proliferative response since UV-inactivated VSV was immunogenic in these in vitro tests. Following primary infection, antigen-specific proliferative responses could be stimulated by several strains of VSV-NJ, but not by VSV-Indiana (VSV-Ind) serotype virus. Secondary infection had relatively little effect on the proliferative response to VSV-NJ strains, but it did cause the PBM to gain responsiveness to VSV-Ind.

Animals↗
PubMed1989

Antigen-specific proliferative responses to vesicular stomatitis virus following immunization of cattle with inactive virus.

Peripheral blood mononuclear cells (PBM) from four normal cows with no known exposure to vesicular stomatitis virus (VSV) were cultured with a New Jersey (NJ) serotype (Ogden) VSV that had been UV-irradiated and inactivated. PBM from these animals produced no detectable proliferative response when incubated with varying concentrations of VSV-NJ (Ogden) ranging from 10 ng to 10 micrograms protein/ml. Two of these cows were immunized with an experimental VSV-NJ vaccine and their PBM were tested at various intervals after immunization. PBM tested 14 days after the initial immunization produced readily detectable antigen-specific proliferative responses when cultured with UV-irradiated strains of VSV-NJ. Following a second immunization, lower concentrations of antigen were sufficient to stimulate the proliferative response and the magnitude of the proliferative response was increased. The responsiveness persisted for at least 6 months after these two immunizations. The specificity of the proliferative response was examined by comparing the responses stimulated by one VSV-Ind and four VSV-NJ serotype strains. The PBM from the immunized cows produced proliferative responses that were essentially specific for the VSV-NJ serotype antigens. In dose titrations, the VSV-NJ antigens were 300-1000-fold more effective than was the VSV-Ind antigen. Thus, persistent antigen-specific proliferative responsiveness that is serotype specific can be stimulated by immunizing cattle with an inactivated VSV vaccine.

Animals↗
PubMed1988

Lysis by RNK-16 cytotoxic lymphocyte granules. Rate assays and conditions to study control of cytolysis.

Dense subcellular granules of cytolytic lymphocytes can mediate rapid lysis of erythrocytes or nucleated cells. The granules contain several different proteases and proteoglycans that regulate cytolysis. We describe a rate assay that we have already used to demonstrate the requirement for serine proteases in granule-mediated lysis. In this assay, 51Cr-labeled erythrocytes are lysed by limiting concentrations of granules from RNK-16 tumor cells. Cytolysis is initiated by the addition of calcium (1 mM final concentration) and stopped at 0.5-1 min intervals by acidification to pH 6.0. The effects of the granule protein concentration, temperature, the concentration of erythrocytes, pH, and the concentration of calcium on the rate of lysis are reported. A preliminary mathematical approach is described and suggested as a means to differentiate 'lag' or activation times from the initial burst of lysis. With this rate assay, we have found four classes of protease inhibitors that block granule-mediated lysis (Hudig et al. (1987) Biochem. Biophys. Res. Commun. 149, 882). The utility of the rate assays is underscored by the observation that reversible protease inhibitors only showed rates of cytolysis whereas irreversible protease inhibitors stopped cytolysis completely. Rate assays are essential for future analyses of the complex physiological regulation of granule-mediated cytotoxicity by proteases, endogenous protease inhibitors and proteoglycans.

Animals↗
PubMed1988

Cyclosporin A does not inhibit the PHA-stimulated increase in intracellular Ca2+ concentration but inhibits the increase in E-rosette receptor (CD2) expression and appearance of interleukin-2 receptors (CD25).

The immunosuppressive drug cyclosporin A (CsA) inhibits mixed lymphocyte responses, blocks the generation of cytotoxic T lymphocytes, and inhibits the T lymphocyte proliferative response stimulated by polyclonal activators such as phytohemagglutinin (PHA). Nevertheless, there have been contradictory reports attempting to explain the mechanism(s) for this immunosuppressive activity. In the current studies, human peripheral blood mononuclear cells (PBM) were stimulated with PHA in the presence or absence of CsA. Flow cytometric examination of PBM loaded with the Ca2+-sensitive dye Indo-1 showed that concentrations of CsA sufficient to inhibit 90-100% of tritiated thymidine incorporation had no effect on the PHA-stimulated increase in the intracellular Ca2+ concentration ([Ca2+]i). Likewise, inhibitory amounts of CsA had virtually no effect on the increase in cell volume that occurs during T lymphocyte activation. These results were not altered by pretreating the PBM with CsA for 30 min at 37 degrees C prior to adding the PHA. On the other hand, inhibitory concentrations of CsA prevented the expression of receptors for T cell growth factor (interleukin-2, IL-2), as measured by monoclonal antibodies to CD25 after 16-24-hr incubation. In like manner, CsA also prevented the increase in the expression of the E-rosette receptor (CD2) on these same cells. If cultures containing PHA and inhibitory amounts of CsA were incubated for 40-72 h, there was partial recovery both of proliferative activity and of the expression of CD25 and CD2. Thus, CsA does not appear to affect the initial activation signal(s), but does interfere with one or more subsequent events necessary to initiate the appearance of "activation antigens."

Antigens, Surface↗
PubMed1988

Identification of inflammatory cells in bovine milk by flow cytometry.

Cells recovered from normal or mastitic bovine milk were examined by flow cytometry. All milk samples contained particulate material that was heterogeneous in size and that produced a right-angle light-scatter signal equal to or greater than that produced by human or bovine neutrophils. Although this material labeled with Hoechst 33342, it produced fluorescence intensities below that of intact bovine cells, suggesting that it consisted of cell fragments. Mastitic milk additionally contained other populations of cells that were poorly resolved from the normal particulate material by size (electronic volume sensor) and right-angle light scatter. In order to improve this resolution, the milk cells were incubated with carboxydimethylfluorescein diacetate (CMFDA) to label intact cells. When milk samples labeled with CMFDA were examined by dual-parameter analysis using green fluorescence and right-angle light scatter, five or more populations of cells could be identified in mastitic milk. These populations included intact and degenerate neutrophils, lymphocytes, including both small and activated cells, monocytes, and large activated macrophages containing many vacuoles and phagocytosed particles. Using this procedure, all the animals in the University of Nevada-Reno Holstein dairy herd were tested once a month for 6 months. In addition, individual animals with mastitis were examined one or more times each day during the course of the inflammatory process. In the routine screening, the flow cytometric examination detected mastitis before overt symptoms developed. In cows identified to have mastitis, the flow cytometric examination provided prognostic information regarding the success of treatments.

Animals↗
PubMed1987

Simultaneous increased expression of E-rosette receptor (CD2, T11) and T cell growth factor receptor on human T lymphocytes during activation.

The E-rosette receptor (CD2, T11) is a differentiation antigen expressed on immature and mature human T lymphocytes. Activation of T cells from human peripheral blood with phytohemagglutinin (PHA) or with monoclonal antibody to the CD3-Ti complex (anti-Leu-4) caused the expression of CD2 to increase 10- to 20-fold. Dual parameter correlated analyses with antibody to the T cell growth factor (TCGF) receptor (anti-Tac) and anti-CD2 antibody demonstrated that the increase in CD2 expression occurred at the same time and on the same cells that expressed the TCGF receptor after stimulation with PHA. The increased expression of CD2 and the initial expression of Tac were totally inhibited by cycloheximide, but were not affected by sufficient actinomycin-D to block the T cell proliferative response. The expression of CD2 was compared with the expression of CD4 and CD8, i.e., T cell differentiation antigens on cytotoxic/suppressor or helper T cells, respectively. Although virtually all of the small percentage of freshly isolated Tac+ peripheral blood cells belonged to the CD4+, CD8- subset, both CD4+ and CD8+ T cells were equivalently activated by PHA to express Tac. By 20-30 hr after activation, the expression of CD4 or CD8 was initially decreased 10-50%. Subsequently, the expression of CD4 and CD8 returned to the levels on resting T cells but did not increase further. Therefore, the increase in CD2 expression does not reflect a universal property of cell surface antigens on activated T lymphocytes.

Antibodies, Monoclonal↗
PubMed1987

Activated neutrophils exhibit enhanced phagocytosis of Cryptococcus neoformans opsonized with normal human serum.

We studied the effect of agents that activate neutrophils on phagocytosis of C. neoformans. The amount of CR3 on the surface of neutrophils was used as a marker for neutrophil activation. Surface CR3 was estimated by flow cytometry using phycoerythrin-labelled anti-CR3 (anti-Leu-15) monoclonal antibody. Phagocytosis was determined by incubation of neutrophils with cryptococci that had been preincubated with normal human serum. We found that treatment of neutrophils with (i) the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine, (ii) zymosan activated serum, (iii) supernatant fluid from a mixed leucocyte culture, or (iv) supernatant fluid from human leucocytes cultured with phytohaemagglutinin produced a dose-dependent increase in CR3 density. These agents also markedly enhanced phagocytosis of opsonized cryptococci in a parallel dose-dependent fashion. These results indicate that phagocytosis of cryptococci opsonized with normal human serum is markedly enhanced by treatment of neutrophils with reagents that stimulate neutrophils. Our results demonstrate that neutrophils activated in an appropriate manner are capable of efficient phagocytosis of encapsulated cryptococci. This potential phagocyte activity may account in part for the high natural resistance to cryptococcosis.

Cryptococcus↗
PubMed1986

The induction of the human T-cell growth factor receptor precedes the production of RNA and occurs in the presence of inhibitors of RNA synthesis.

The receptor for T-cell growth factor (TCGF) is an activation antigen that is present in low amounts on a small fraction of resting T lymphocytes. The TCGF receptor on human T cells can be detected with the anti-Tac monoclonal antibody within 7-12 h of stimulating the cells with phytohemagglutinin (PHA). In the current studies, we examined human lymphocytes cultured alone, with PHA, or with PHA plus sufficient actinomycin-D to inhibit RNA synthesis. After varying intervals, aliquots of the lymphocytes were stained with acridine orange (AO) or pyronin-Y(PY) to measure RNA and/or with anti-Tac plus FITC goat anti-mouse Ig. Tac expression began to increase after 6-8 h incubation with PHA, whereas increases in PY or AO staining were not detected until 12 h or later. Furthermore, the initial increase in Tac expression was not affected by sufficient actinomycin-D to block all detectable nucleic acid synthesis. Therefore, it appears that the initial expression of TCGF receptors detected after lymphocyte activation does not require de novo production of RNA.

Antibodies, Monoclonal↗