Cell Blot Assay Research Articles

Single-cell analysis of dihydroartemisinin–induced apoptosis through reactive oxygen species–mediated caspase-8 activation and mitochondrial pathway in ASTC-a-1 cells using fluorescence imaging techniques

Dihydroartemisinin (DHA), a front-line antimalarial herbal compound, has been shown to possess promising anticancer activity with low toxicity. We have previously reported that DHA induced caspase-3-dependent apoptosis in human lung adenocarcinoma cells. However, the cellular target and molecular mechanism of DHA-induced apoptosis is still poorly defined. We use confocal fluorescence microscopy imaging, fluorescence resonance energy transfer, and fluorescence recovery after photobleaching techniques to explore the roles of DHA-elicited reactive oxygen species (ROS) in the DHA-induced Bcl-2 family proteins activation, mitochondrial dysfunction, caspase cascade, and cell death. Cell Counting Kit-8 assay and flow cytometry analysis showed that DHA induced ROS-mediated apoptosis. Confocal imaging analysis in a single living cell and Western blot assay showed that DHA triggered ROS-dependent Bax translocation, mitochondrial membrane depolarization, alteration of mitochondrial morphology, cytochrome c release, caspase-9, caspase-8, and caspase-3 activation, indicating the coexistence of ROS-mediated mitochondrial and death receptor pathway. Collectively, our findings demonstrate for the first time that DHA induces cell apoptosis by triggering ROS-mediated caspase-8/Bid activation and the mitochondrial pathway, which provides some novel insights into the application of DHA as a potential anticancer drug and a new therapeutic strategy by targeting ROS signaling in lung adenocarcinoma therapy in the future.

Cosecretion of Prolactin and Growth Hormone by Dispersed Pituitary Cells of the Adult Bullfrog, Rana catesbeiana

The coexistence of prolactin (PRL) and growth hormone (GH) was previously demonstrated in newly hatched bullfrog (Rana catesbeiana) tadpoles, whereas in adult bullfrogs, there were no cells containing both PRL and GH. However, a cell blot assay with enzymatically dispersed adult pituitary cells demonstrated the existence of cells secreting both PRL and GH. The number of cells secreting both PRL and GH was reduced by a protein synthesis inhibitor, cycloheximide, but not by an RNA synthesis inhibitor, actinomycin D. In situ hybridization and immunostaining of intact pituitary glands revealed the existence of GH mRNA in some of the PRL-immunoreactive cells and of PRL mRNA in some of the GH-immunoreactive cells. We propose that dispersion of the pituitary cells triggered the translation of GH mRNA in the PRL cells and/or of PRL mRNA in the GH cells.

Direct effects of growth hormone (GH)-releasing hexapeptide (GHRP-6) and GH-releasing factor (GRF) on GH secretion from cultured porcine somatotropes

Growth hormone (GH)-releasing hexapeptide (GHRP-6) belongs to the expanding family of synthetic GH secretagogues (GHSs). Previous studies have shown that non-peptidyl GHRP-6 analogues stimulate GH release in vivo in pigs, and interact synergistically with GH-releasing factor (GRF), but its direct effects on porcine somatotropes have not been addressed hitherto. In the present study, we have evaluated the response of cultured porcine pituitary cells to GHRP-6, and its interaction with GRF and somatostatin (SRIF). Secretory response of somatotropes was assessed by using two distinct techniques. GH released by monolayer cell cultures was evaluated by enzyme immunoassay, whereas that secreted by individual somatotropes was measured by immunodensitometry using a cell blotting assay. Our results demonstrate that both GHRP-6 and GRF stimulated GH release from monolayer cultures at doses equal to or above 10 −9 M. Use of cell immunoblot assay demonstrated that, like GRF, the hexapeptide acts directly upon porcine somatotropes to exert its action. Moreover, regardless of the technique applied, combined administration of GHRP-6 (10 −6 or 10 −9 M) and GRF (10 −8 M) resulted in an additive, but not synergistic, stimulatory GH response. Finally, SRIF (10 −7 M) inhibited the stimulatory effect of GHRP-6 alone or in combination with GRF. These results indicate that GHRP-6 directly and effectively stimulates GH secretion from porcine somatotropes in vitro, and acts additively when coadministered with GRF. Therefore, the synergistic stimulatory effect of GHSs and GRF reported in vivo in this species might require additional factors that are lacking in the in vitro situation.

Arbutin increases the pigmentation of cultured human melanocytes through mechanisms other than the induction of tyrosinase activity.

We assessed the effects of arbutin on the pigmentation of cultured normal human melanocytes. As indicated by a cell-blotting assay, arbutin at concentrations in the range of 0.5-8 mM increased the pigmentation of the cultured melanocytes, while kojic acid at concentrations in the range of 0.5-4 mM decreased the pigmentation. The pigmentation-augmenting effect of arbutin was further confirmed by the results of a cell-pelleting assay, the traditional method of assessment. Treatment of the cells with arbutin increased the melanin content of the cells and the protein content as well. On the other hand, the tyrosinase activity in the cells was reduced by arbutin treatment. The levels of transcription of tyrosinase and tyrosinase related protein-1 genes were not affected by arbutin treatment as indicated by a semi-quantitative reverse transcription-polymerase chain reaction assay. These results demonstrate that arbutin promotes an increase in pigmentation of cultured human melanocytes that is not mediated by augmented tyrosinase activity.

Identification of an immunogenic histone-like protein (HLPMt) ofMycobacterium tuberculosis

We report the identification of the first histone-like protein ofMycobacterium tuberculosis(MTB) (HLPMt). The T cell blot assay was used to identify antigens of MTB associated with human immune response in healthy contacts. Fraction 21 corresponding to proteins in the molecular weight range of approximately 30 kDa were found to be immunogenic in tuberculin reactors. None of the fractions were found to be immunogenic by this assay in non-reactors to tuberculin. All sera, irrespective of the source, showed reactivity with MTB antigen(s) over a wide molecular weight range (205 16 kDa). In the present study fraction 21 was processed for the generation of murine polyclonal sera and amino acid sequencing. The sequence of a 16-amino acid long peptide showed a 100% homology with an open reading frame (ORF) in the translated sequence of cosmid cY349 (Sanger Centre, Cambridge, UK). The ORF was predicted to code for a protein of 214 amino acids. Oligonucleotide primers were synthesized based on the nucleotide sequence located at the 5′ and 3′ regions of the gene. The gene encoding the predicted protein was PCR-amplified, cloned, sequenced and expressed inEscherichia colias a protein of 28 kDa. The expressed HLPMtprotein was shown to react with the polyclonal murine sera originally raised against fraction 21. Human immune response to the recombinant HLPMtprotein was demonstrated by its ability to induce lymphoproliferation in peripheral blood derived mononuclear cells, and the presence of anti-HLPMtantibodies in pooled patient sera by immunoblot. The recombinant HLPMtprotein elicited a vigorous lymphoproliferative response especially in healthy tuberculin reactors compared to non-reactors and patients of tuberculosis, (P⪡ 0.05). The protein has unique dual domains with homology to both bacterial histone-like proteins (HU) and eukaryotic histone H1. Homology to prokaryotic and eukaryotic deoxyribonucleic acid (DNA)-binding proteins suggested that HLPMtcould bind DNA. DNA-binding properties were confirmed by South-Western analysis strongly suggesting an interaction between HLPMtand the MTB chromosome.

DNA binding protein of mycobacteria and human immune response

A dual step procedure was used to identify a 30 kDa DNA binding protein of mycobacteria (HLPMt) which is a target of human T and B cell response. The immunodominance of HLPMt was estabished by T cell blot assay as well as by subtractive immunoblot assay. This protein is not secreted into the extracellular culture fluid and is different from the 85 ABC complex of proteins as seen by immunoblots and ELISA. The protein is capable of inducingin vitro lymphoproliferation in tuberculin reactors. The protein was purified for the generation of monospecific sera and for amino acid sequencing. The sequence of the 16 amino acid long peptide derived from the 30 kDa protein showed a 100% homology with the translated sequence of a cosmid cY349 (Sanger Centre, Cambridge, UK). The ORF was predicted to code for a protein of 214 amino acids. Oligonucleotide primers were designed against the 5' and 3' end of the gene and the gene was PCR amplified, cloned and expressed inE.coli. The protein has unique dual domains which show homology to both bacterial HU proteins and to eukaryotic histones H1.

CD5+ B lymphocytes are the main source of antibodies reactive with non‐parasite antigens in Trypanosoma congolense‐infected cattle

Mice infected with African trypanosomes produce exceptionally large amounts of serum IgM, a major part of which binds to non-trypanosome antigens such as trinitrophenol and single-strand DNA. In this paper, we describe that in cattle infected with Trypanosoma congolense and T. vivax, similar antibodies are found, although they bind mainly to protein antigens, such as beta-galactosidase, ovalbumin and ferritin. The parasite non-specific IgM antibodies appear around the same time as the parasite-specific antibodies, but their origin and function are not clear. We tested the hypothesis that CD5+ B cells (or B-1 cells), which increase during trypanosome infections in cattle, are responsible for production of antibodies to non-trypanosome antigens. Splenic CD5+ and CD5- B cells from infected cattle were sorted and tested in a single cell blot assay. The numbers of immunoglobulin-secreting cells were similar in both B-cell populations. However, antibodies with reactivity for non-trypanosome antigens were significantly more prevalent in the CD5+ B-cell fraction and were exclusively IgM. The preference for production of these antibodies by CD5+ B cells and the expansion of this subpopulation during infections in cattle, strongly suggest that CD5+ B cells are the main source of trypanosome non-specific antibodies. We propose that these antibodies are natural, polyreactive antibodies that are predominantly secreted by CD5+ B cells. Since B-1 cells are up-regulated in many states of immune insufficiency, the immunosuppression associated with trypanosome infections may be responsible for the increase of this subset and the concomitant increase in trypanosome non-specific antibodies.

A simple and quantitative cell-blotting assay for evaluation of pigmentation of cultured melanocytes and its use in demonstrating the depigmenting effect of lactoferrin.

A simple and quantitative cell-blotting assay for evaluation of pigmentation of cultured melanocytes and its use in demonstrating the depigmenting effect of lactoferrin.

Regulation of bFGF expression and ANG II secretion in cardiac myocytes and microvascular endothelial cells.

Basic fibroblast growth factor (bFGF; fibroblast growth factor-2) and angiotensin II (ANG II), among other peptide signaling autacoids (cytokines), are known to regulate the phenotypic adaptation of cardiac muscle to physiological stress. The cell type(s) in cardiac muscle responsible for ANG II synthesis and secretion and the role of endogenous cytokines in the regulation of bFGF induction remain unclear. With the use of confluent, serum-starved, low-passage cultures of cardiac microvascular endothelial cells (CMEC), ANG II could be detected in cellular lysates and in medium conditioned by these cells with the use of high-performance liquid chromatography followed by radioimmunoassay. The secretion of angiotensins by individual CMEC could be detected with a cell-blot assay technique. ANG II secretion was decreased by brefeldin A, an agent that interrupts constitutive and regulated secretory pathways for peptide autacoid/ hormone synthesis, suggesting de novo synthesis, activation, and secretion of angiotensins by CMEC. In primary isolates of adult rat ventricular myocytes (ARVM) and CMEC, ANG II, acting at ANG II type 1 receptors in both cell types, was found to increase bFGF mRNA levels measured by ribonuclease protection assay. Endothelin-1 (ET-1), which is known to be synthesized by CMEC, and bFGF itself, which has been detected in both ARVM and CMEC, increased bFGF transcript levels in both cell types. Interleukin-1beta (IL-1beta), which like ANG II and ET-1 is known to activate mitogen-activated protein kinases in both ARVM and CMEC, increased bFGF mRNA levels only in cardiac myocytes. Thus cytokines such as ANG II, ET-1, bFGF, and IL-1beta locally generated by cellular constituents of cardiac muscle, including CMEC, regulate bFGF mRNA levels in a cell type-specific manner.

Galanin Inhibits ACTH Releasein Vitroand Can Be Demonstrated Immunocytochemically in Dispersed Corticotrophs

Double labeling of dispersed anterior pituitary cells revealed the coexistence of galanin immunoreactivity with adrenocorticotropic hormone (ACTH) as well as prolactin. However, only laser confocal microscopy showed three different areas of immunoreactivity within the corticotroph cytoplasm, two of them for ACTH and galanin separately and the third containing both immunoreactivities. To determine a possible relation between ACTH cells and galanin, 4-day cultures of anterior pituitary cells from female rats were examined by cell blot assay, and they showed ACTH release inhibition by 10−6Mgalanin. Furthermore, after 17 β-estradiol treatment to maximize lactotroph galanin releasein vitro,the cell blotting-assessed secretory level of corticotroph cells was very similar to that of cells in the presence of 10−6Mgalanin. In fact, immunoneutralization with galanin antiserum quenched the inhibitory effect of galanin on ACTH secretion. Our study suggests that locally produced galanin can modulate corticotropin release.

Non-muscle myosin II heavy chain has a cryptic cell-adhesion domain

We have discovered a cryptic cell-adhesion domain in non-muscle myosin II heavy chain. A 205 kDa cell-adhesion-promoting polypeptide (p205) was extracted from BHK cells by Nonidet P-40 or Dounce homogenization. Adhesion to p205 was specifically inhibited by the peptide Gly-Arg-Gly-Asp-Ser-Pro, indicating a role for the Arg-Gly-Asp cell-adhesion motif. Purified p205 was identified as non-muscle myosin II heavy chain, based on sequence analysis and on the cross-reactivity of p205 with anti-(bovine trachea myosin) antibodies. Further experiments showed that the heavy chain of purified myosin II has cell-adhesion-promoting activity in a cell-blotting assay, and cross-reacted with anti-p205 antibodies. Finally, the adhesion domain was located in the tail portion of myosin II heavy chain, where an Arg-Gly-Asp-containing sequence can be found.

Cell adhesion to proteins separated by lithium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto a polyvinylidene difluoride membrane: a new cell-blotting technique

Cell blotting, although conceptually simple, has failed to achieve wide practical application. Described here is a new cell-blotting technique which involves cell adhesion to protein bands after separation by lithium dodecyl sulfate-polyacrylamide gel electrophoresis (LDS-PAGE) and blotting onto polyvinylidene difluoride (PVDF) membrane at 4°C. Cell bands adherent on PVDF are detected using hematoxylin, or propidium iodide (PI) staining followed by viewing under ultraviolet (UV) light. The technique allows quick microscopic visualization of adherent cells composing the bands, without requiring clearing of the membrane. Representative cell adhesion proteins from different sources, i.e., plant lectins (e.g., phytohemagglutinin, PHA; concanavalin A, ConA; and wheat germ agglutinin, WGA); extracellular matrix (ECM) proteins; and integral membrane proteins (e.g., recombinant soluble vascular cell adhesion molecule-1, rs VCAM-1) were tested for cell binding by the new cell-blotting technique using human lymphoid progenitor (NALM-6) and myeloid progenitor (KG1a) cell lines. Cell adhesion proteins retained their adhesion function in all cases tested. Specificity of cell binding onPVDF blot was demonstrated by inhibition of cell adhesion to WGA protein bands using an appropriate sugar, i.e., N-acetyl D-glucosamine. The cell blotting assay was comparable in sensitivity to Coomassie blue staining of protein bands. The ability to conduct protein extraction, separation and blotting at low temperature avoids thermal denaturation, thereby preserving the adhesion properties of the proteins. The electrophoretic/blotting system has unique detergent removal/protein renaturation properties and the ability to preserve functionally active adhesion protein complexes. The cell-blotting technique described is sufficiently robust for routine application in the investigation of novel cell adhesion proteins.

The cell blot assay in analysis of rat anterior pituitary cell secretion

We have investigated the efficacy of the cell blot assay in analysis of the secretion of hormones and peptides from rat anterior pituitary cells. The dissociated cells are cultured on pieces of translucent polyvinylidene difluoride membrane, on which their secretory products are adsorbed and subsequently immunostained. The area and integrated optical density of the stained 'halo' surrounding individual cells is measured by microscopical image processing and the values for basal secretion of a particular hormone or peptide are compared with those after application of secretagogues or inhibitors. Our experiments tested established responses of dissociated rat anterior pituitary cells; in general, the results were as expected. Double immunoenzymatic staining could be used to show secretion of two products from the same or different cells in one preparation, and immunofluorescence with fluorescein- and/or rhodamine-labelled antibodies could be used instead of enzyme-linked immunolabelling. Optimal dilutions of immunoreagents were much higher than those used for immunocytochemistry on tissue sections. Although the cell blot assay does not provide absolute quantification, since some of the secreted product escapes into the medium, it is a relatively easy and economical way for morphologists to compare secretion from individual cells under varying conditions.

Synthesis and release of calcitonin-like immunoreactivity by anterior pituitary cells: evidence for a role in paracrine regulation of prolactin secretion.

Calcitonin (CT) is a potent and specific inhibitor of basal and TRH-induced PRL release and PRL mRNA levels in rat anterior pituitary (AP) cells, an action mediated through specific inhibition of the Ca(2+)-inositol phosphate messenger system. Because CT and CT-like peptides have been reported to be present in the AP, the present studies investigated 1) whether 35S-labeled CT-like substances can be precipitated from rat AP cell lysates, 2) whether immunoreactive CT is secreted from rat AP cells, as assessed from a cell blot assay and from RIA of spent medium from cultured AP cells, and 3) whether the release of PRL from cultured rat AP cells can be influenced by immunoneutralization of endogenous CT. Dispersed rat AP cells were labeled with [35S]cysteine, CT-like substances were immunoprecipitated from the lysate with antihuman CT (anti-hCT) and antisalmon CT (anti-sCT) immunoglobulin G conjugated to protein-A-Sepharose beads, and the immunoprecipitates were fractionated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by fluorography. The results revealed that both anti-sCT and anti-hCT sera precipitated three major bands of 35S-labeled material from AP cell lysates, with approximate molecular masses of 23, 6.5, and 3.5 kilodaltons. Synthetic sCT completely displaced anti-sCT-precipitable bands and partially displaced anti-hCT precipitates. The cell blot assay revealed the presence of CT-immunopositive AP cells, and secretion of this substance was indicated by zones of secretion surrounding these cells. Immunoreactive CT was also detected in spent medium from cultured rat AP cells, and the average rate of release over 4 days of culture was approximately 1 ng sCT eq/3 million AP cells.24 h. Both anti-sCT and anti-hCT sera significantly stimulated PRL release from rat AP cells, and the stimulatory effect of anti-sCT serum was dose dependent up to a concentration of 1:50. The present findings demonstrate that CT-like peptide(s) is synthesized and released from cultured AP cells and suggest that this peptide may participate in the regulation of PRL secretion via paracrine or autocrine actions.

Application of an optimized immunostaining technique to evaluate the heterogeneous secretory response from porcine somatotropes by cell blotting.

The objective of the present study was to quantify the absolute hormone release from individual porcine pituitary cells incubated on polyvinylidene difluoride (PVDF) transfer membranes (cell-blot assay). After immunoperoxidase staining, growth hormone (GH) release from isolated somatotrope cells appeared like a colored zone of secretion surrounding the cell. Optical densities of these secretion zones were quantitated by computerized image analysis and translated into picograms by means of an appropriate standard curve. As a prior step, the staining method and the optimal immunocytochemical conditions were selected by applying purified porcine growth hormone (pGH) to the transfer membranes. The avidin-biotin-peroxidase nickel-intensified (ABC-Ni) method produced a better resolution than the peroxide-anti-peroxidase (PAP) method, although both techniques were similar with regard to background, sensitivity, and range of quantitation. The amount of GH released from single porcine somatotropes was highly heterogeneous, although the cells were treated under the same conditions. Moreover, this fact was consistent with the stimulation of the average release of GH by GH-releasing factor (GHRF) but not by GHRF+somatostatin (SRIF). Our results confirm the availability of the recently developed cell-blot assay and support the concept of functional heterogeneity in anterior pituitary cell populations.

Effect of training on insulin secretion from single pancreatic beta cells

The objective of this study was to determine whether insulin secretion from single pancreatic beta cells is reduced by endurance training. Male Sprague Dawley rats either trained (T, N = 9) for 11 wk on a rodent treadmill, remained sedentary, and were fed ad libitum (S, N = 8) or remained sedentary and were food restricted (pair fed, PF, N = 8) so that final body weights were similar to T. After training, T had significantly higher red gastrocnemius muscle citrate synthase activity compared with S and PF. In vivo insulin secretion was lower in T (4.6 +/- 1.4 ng.ml-1, mean +/- SEM of 70' + 90' concentrations during a hyperglycemic glucose clamp) when compared with S, 8.1 +/- 1.6 and PF, 9.7 +/- 1.7 ng.ml-1. In vitro insulin secretion from single beta cells was measured using the cell blot assay (Kendall and Hymer, Endocrinology, 121:2260-2262, 1987) and T, had lower secretion 2.6 +/- 1.4 pg.cell-1 when compared (P less than 0.05) with S, 6.7 +/- 0.6, but not lower than PF, 4.1 +/- 1.7 pg.cell-1. These data suggest that some of the training-induced reduction in insulin secretory response to glucose may be attributable to changes within the beta cell itself.

Rapid, simple identification of individual osteoblastic cells and their specific products by cell blotting assay

Biochemical and molecular biological studies of osteoblastic cell function and hormonal regulation are frequently confounded by the inherent cellular heterogeneity and phenotypic instability of existing in vitro and in vivo model systems. A new technique (derived from Western blotting or antibody-based detection of protein molecules bound to nitrocellulose paper) is described for identification of individual cells which synthesize osteoblast-specific gene products (bone Gla-protein, type I collagen, and alkaline phosphatase) or produce cAMP in response to parathyroid hormone (PTH) or isoproterenol. Dispersed primary neonatal rat calvariae or osteogenic sarcoma cells were "plated" on Immobilon-P (a hydrophobic transfer membrane with very high protein-binding capacity) for 30 minutes to several hours, followed by agonist treatment, formalin fixation, hematoxylin staining, and immunostaining with a battery of antibodies specific for osteoblastic products. Individual cells and their secretory zones were visualized by light microscopy and counted. Treatment with PTH with or without isoproterenol resulted in increases in the percentages of osteoblastic cells elaborating cAMP, as well as the intensity of immunostaining, but had no effects on MCF-7 cells, a nonosteoblastic breast carcinoma control line. The percentage of cells within each primary osteoblastic cell population isolated or rat osteogenic sarcoma cell clone (G2 or C12) that elaborated bone-specific proteins or that generated cAMP in response to PTH varied with time and the individual cellular preparation, reconfirming the cellular heterogeneity of these systems. This method, in conjunction with techniques such as in vitro hybridization, should prove useful in characterizing discrete osteoblastic bone cell subpopulations and in clarifying mechanisms of hormonal regulation by local and systemic agents.

Demonstration of IL-1α and IL-1β secretion by the monocytic leukemia cell line, THP-1

This study examined the secretion of IL-1α and IL-1ß by THP-1 leukemia cells following activation with mezerein and promotion of synthesis by interferon (IFN-γ). Interleukin-1 (IL-1) was not detected by co-mitogenic thymocyte assays of crude supernates. Isoelectrofocusing of concentrated medium showed that all biologically active IL-1 migrated at a pH of 6.8–7.2, indicating that the major secreted form was IL-1β. Double antibody ELISA confirmed the presence of IL-1β, but failed to detect IL-1α in isofocused fractions. Although it appeared that THP-1 cells do not secrete IL-1α; an inhibitor of thymocyte response to IL-1 was present in conditioned medium, migrated in an acidic pH range and masked the expression of biologically active rIL-1α and rIL-1ß. In contrast, IL-1α was detected using a cell blotting assay. This technique permitted visualization of subpicogram levels of IL-1 when secreted by cells attached to an immunoblotting paper. Cell blotting showed that a greater proportion of attached cells incubated for 24 h in medium containing mezerein and IFN-γ secreted IL-1 than cells in control medium. In conclusion, the amount of immunoreactive or biologically active IL-1α secreted by stimulated THP-1 cells appeared to be much lower than that reported for human peripheral blood monocytes.

Measurement of hormone secretion from individual cells by cell blot assay

Publisher Summary This chapter discusses the measurement of hormone secretion from individual cells by cell blot assay. In the cell blot assay, cells are incubated on, and bound to, a transfer membrane. Hormone released from the cells is captured on the membrane. The membrane is then processed according to Western blot methodology. This includes incubation in primary antiserum, secondary antiserum to which an enzyme is conjugated, and enzyme substrate. The released hormone product surrounding the hormone-secreting cells (e.g., prolactin and growth hormone) becomes visible as a zone of secretion immediately encompassing the cell. This secretion zone is then analyzed and quantified. Heterogeneity of hormone release per cell becomes apparent. One of the limitations of the cell blot assay is the inability to visualize the nonsecreting cells on the immobilon membrane. Advantages of the cell blot assay include its simplicity and rapidity, use of dilute preparations of antisera, objectivity in measurement of hormone secretion, and capability of quantification of hormone release from single cells.

Heterogeneity in mammotrophs prepared from diethylstilbestrol-induced prolactinomas.

Pituitary glands from ovariectomized F-344 rats bearing diethylstilbestrol-containing capsules for 70-100 days were dissociated and separated into three size classes by unit gravity sedimentation. The average percentage of large mammotrophs in fraction I was 86 +/- (SE) 2%, the percentage of intermediate-sized mammotrophs in fraction II was 86 +/- 4%, and the percentage of small mammotrophs in fraction III was 72 +/- 3%. By electron microscopy, cells in fractions I and II contained cytoplasmic secretory granules, whereas those in fraction III were either sparsely granulated or agranular. Cells retained their morphology after culture. Biological (B) and immunological (I) assays of PRL released into the medium after 24-h culture both showed that fraction I and II cells released more hormone than fraction III cells. The B/I ratios of released PRL were consistently greater than 1. Analysis of the separated mammotrophs by flow cytometry indicated major differences in light scatter profiles, both before and after culture. Since these differences have been shown to reflect secretory granule content, changes in perpendicular light scatter after culture were interpreted to indicate that small mammotrophs accumulated granules, whereas some of the large mammotrophs lost granules. Results from a cell blot assay indicated that fraction I and II cells secreted more hormone than fraction III cells. This same rank order was found when live cells were stained and analyzed for surface PRL. However, the reverse plaque assay yielded different information, viz. more plaques were found around fraction III cells than in fraction I or II cells. Finally, PRL contained in alkaline extracts of fraction I, II, and III cells was analyzed by Western blotting after electrophoresis under nondenaturing, denaturing, or denaturing and reducing conditions. Fraction III cells contained a prominent and unique PRL variant that had low mobility in native gels and an apparent mol wt in the range of 10-14K. After 24 h the culture medium from fraction III cells also contained this low mol wt variant. Our data indicate considerable heterogeneity in form and function within the mammotroph population of diethylstilbestrol-induced prolactinomas.