After fertilization, sperm surface components remain as a patch in sea urchin and mouse embryos

Sea urchin and mouse sperm that are labeled on their surfaces with fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TMRTC) or 125I-diiodofluorescein isothiocyanate (125IFC) remain viable and can fertilize eggs. When sea urchin eggs were fertilized with 125IFC-labeled sperm, the radioactivity from the sperm was quantitatively transferred to the egg (at a ratio of one sperm equivalent per egg) and persisted in the embryo as it developed to the pluteus larval state (5 days at 12 degrees C). The radioactivity was acid-precipitable and was associated with the particulate fraction of embryo homogenates. In addition, FITC-labeled sea urchin sperm were used to fertilize eggs, and the labeled components were followed by fluorescence microscopy. In the embryo, labeled sperm components were present as a discrete patch that was partitioned unequally during early cleavages. In experiments using mouse sperm labeled with TMRTC, the labeled sperm components were also transferred to the embryo as a discrete patch that was again distributed unequally after cleavage. This physiological cell fusion system therefore has distinctive characteristics: there is limited lateral mobility of surface components, which have a low turnover rate unlike that see in other systems. In this paper, we discussed the possible morphogenetic role of this unusual behavior.     

CELL LOSS AND INFLUX OF LABELED HOST CELLS IN THREE TRANSPLANTABLE MOUSE TUMORS USING [125I]UdR RELEASE

The [¹²⁵I]UdR loss technique was used to estimate cell loss from RIF-1, EMT6 and KHJJ tumors in order to determine the length of the delay between labeling and the beginning of the loss of labeled cells, and also to calculate a value for ø, the cell loss factor. To determine the importance of reutilization of label released from the gut and/or the influx of labeled host cells, the blood flow to some tumors was occluded during and for 30 min after injection of the label. Relatively small amounts of radioactivity entered occluded RIF-1 tumors during 9 days after injection of [¹²⁵I]UdR, indicating that reutilization of systemic label and influx of labeled host cells are not significant in this system. In contrast, substantial amounts of radioactivity entered occluded EMT6 and KHJJ tumors, reaching 40% of the total activity in non-occluded tumors during 6 days following injection. After corrections were made for this influx of label, the [¹²⁵I]UdR loss curves from RIF-1 and EMT6 tumors were essentially exponential from the first day following injection of label. This was interpreted as indicating the loss of proliferating as well as non-proliferating cells from both tumors. The cell loss factor derived from the [¹²⁵I]UdR loss curves corrected for influx appeared to agree well with published values derived from analysis of percent labeled mitoses curves. In contrast, the corrected [¹²⁵I]UdR loss curves from KHJJ tumors showed that loss of activity began three days after injection of label, indicating that primarily nonproliferating cells are lost from this tumor.     

The fluorescein-mediated interaction of bovine serum albumin with fluorescent derivatives of prolactin and other polypeptides in polarization of fluorescence based assays

During the course of studies relating to the interaction of bovine prolactin with its receptor, it was observed that the fluorescence polarization of prolactin labeled with fluorescein isothiocyanate (fluorescein prolactin) increased from 0.10 to 0.15 upon the addition of bovine serum albumin. Dilution titration measurements show an apparent K_dissociation for the BSA-fluorescein-prolactin complex of 1.1 × 10^?7 M. The stoichiometry of the complex was shown to be approximately 2 mol of fluorescein-prolactin per mole of BSA. The fluorescence emission spectra of the fluorescein moiety in the fluorescein-prolactin is slightly red shifted and increased in intensity in the presence of BSA. The interaction between prolactin and BSA is dependent on the fluorescein attached to the prolactin since [¹²⁵I]prolactin does not form a complex with BSA under identical conditions. The fluorescence polarization of fluorescein-labeled growth hormone and α-lactalbumin also increased in the presence of BSA, suggesting that BSA may interact generally with fluorescein-labeled proteins to form complexes bridged through the fluorescein moiety.     

Lactoperoxidase-catalyzed iodination of surface membrane lipids

Lactoperoxidase-catalyzed iodination of intact cells, is known to label predominantly, if not exclusively, the exposed tyrosine residues of cell surface proteins. The present study demonstrates that during this iodination process surface membrane lipids are also iodinated through an enzyme-dependent step. Phosphatidylcholine, cholesterol-phosphatidylcholine liposomes and confluent secondary cultures of chick embryo cells were iodinated by the lactoperoxidase-glucose oxidase-glucose [¹²⁵I] procedure. Liposomes were efficiently labeled. In the cells, 20–30% of the radioactivity was found in proteins and 20–30% in the lipids. Both neutral and polar lipids were found to bind [¹²⁵I] covalently. Controls in which lactoperoxidase was omitted showed < 6% of the radioactivity found in liposomes or cells labeled with the enzyme.     

Studies on growth inhibition by lectins of penicillia and aspergilli

It has previously been shown in our laboratory that wheat germ agglutinin (WGA) binds to Trichoderma viride and inhibits growth of this fungus. Here we report on the effect of WGA, soybean agglutinin (SBA) and peanut agglutinin (PNA) on Penicillia and Aspergilli. Binding of the lectins to the fungi was examined with the aid of their fluorescein isothiocyanate (FITC) conjugated derivatives. FITC-WGA bound to young hyphal walls of all species, in particular to the hyphal tips and septa, in agreement with the chitinous composition of the cell walls of the two genera. Hyphae of all species examined were labelled, though in different patterns, by FITC-SBA and FITC-PNA, suggesting the presence of galactose residues on their surfaces. Young conidiophores, metulae (of the Penicillia), vesicles (of the Aspergilli), sterigmata and young spores, were also labelled. The three lectins inhibited incorporation of [³H]acetate, N-acetyl-D-[³H]glucosamine and D-[¹⁴C]galactose into young hyphae of Aspergillus ochraceus, indicating interference with fungal growth. Inhibition of spore germination by the three lectins was also observed. Preincubation of the lectins with their specific saccharide inhibitors prevented binding and the inhibitory effects. We conclude that lectins are useful tools for the study of fungal cell surfaces, and may also serve as an important aid in fungal classification. The present findings also support the suggestion that one role of lectins in plants is protection against fungal pathogens.Abbreviations Con A concanavalin A - PNA peanut agglutinin - SBA soybean agglutinin - WGA wheat germ agglutinin - FITC fluorescein isothiocyanate - GlcNAc N-acetyl-D-glucosamine - GalNAc N-acetyl-D-galactosamine     

Preparation of 125I-labeled secretin of high specific radioactivity.

A simple and rapid method of preparing ¹²⁵I-labeled secretin of high specific radioactivity has been developed. Synthetic porcine secretin was iodinated with Na[¹²⁵I] by a modification of the chloramine T method. Purification and separation of labeled from unlabeled secretin was achieved by chromatography on SP-Sephadex C-25 column. The labeled secretin possessed specific radioactivity as high as 500–550 μCi/μg.     

A radiotracer method to study efflux transport of iodide liberated from thyroid hormones via deiodination metabolism in the brain

AimsThyroid hormones (TH) play an important role in the development and functional maintenance of the central nervous system. The purpose of this study was to develop a radiotracer method for studying the in vivo efflux transport of iodide liberated by the TH metabolism in the brain. The rationale of our method is as follows: a radioiodinated compound can enter the brain and rapidly release iodide in situ; the iodide efflux rate can be estimated from the clearance of brain radioactivity after disappearance of the iodinated compound.Main methods6-[¹²⁵I]Iodo-9-pentylpurine ([¹²⁵I]9Pe6IP) was designed to enter the brain and release ¹²⁵I^? by the reaction with glutathione and synthesized from the corresponding bromo derivative in a Br/¹²⁵I exchange reaction. The brain kinetics of radioactivity and radioactive metabolites were investigated after intravenous injection of [¹²⁵I]9Pe6IP into mice. The iodide efflux rate was estimated in mice pretreated with perchlorate, an inhibitor of iodide transport from the brain.Key findingsHigh brain uptake (5.3% injected dose/g) was observed at 1 min, and almost complete conversion of [¹²⁵I]9Pe6IP to ¹²⁵I^? occurred 10 min after injection. The ¹²⁵I^? uptake from the blood was negligible. ¹²⁵I^? was eliminated from the brain along a single-exponential curve with a half-life of 6.0 min. Furthermore, dose-dependent inhibition of ¹²⁵I^? efflux was observed in mice pretreated with perchlorate.SignificanceWe conclude that 9Pe6IP labeled with ¹²⁴I (positron emitter) or ¹²³I (single-photon emitter) may be useful for studying the in vivo efflux transport of iodide in the brain using nuclear medicine imaging devices.     

Fluorescent deacetylcolchicine: New aspects of its activity and localization in PtK-1 cells

PtK-1 cells have been used to localize structures which are decorated by deacetylcolchicine conjugated with fluorescein isothiocyanate (FDC). This colchicine derivative competitively inhibits [³H]colchicine binding to soluble tubulin, is able to depolymerize microtubules (MTs) assembled in vitro, and inhibits cell growth by arresting colchicine-sensitive cells in mitosis. Fixed cells were incubated with FDC (10^?5 M), and/or with tetramethyl rhodamine-labelled anti-tubulin antibodies using the indirect immunofluorescence technique. In double label microscopy the cells show corresponding fluorescence of MTs after incubation with anti-tubulin antibodies and FDC. This can be demonstrated most clearly with cells in mitosis which are preincubated with the microtubule-stabilizing agent taxol. FDC binding is decreased, but not completely blocked, by preincubation of fixed cells with unlabelled colchicine (10^?4 M). Trimethyl colchicinic acid (TMCA) conjugated with FITC, a colchicine derivative which does not bind to soluble tubulin, and FITC alone, inactivated by methylamine, do not bind to MTs. These results demonstrate that FDC is able to decorate and to depolymerize sensitive MTs, indicating a direct action of the drug on these structures. Furthermore, the labelling of other cellular structures by FDC, possibly membrane systems as well as chromatin, is compared with recent data.     

Autoradiographic localizatio of [125I]-C-ANP compared to [125I]-ANP in rat tissue

Summary Whole-body autoradiography demonstrated the different distribution of [¹²⁵I]-C-ANP and [¹²⁵I]-ANP to rat tissues. Highest enrichment of radioactivity of both labelled peptides was found in the kidney. In some organs we found remarkable differences between [¹²⁵I]-ANP and [¹²⁵I]-C-ANP. In the kidney cortex, especially in the glomeruli, as well as in the endocardium, the zona glomerulosa and the medulla of the adrenal gland, where high levels of radioactivity after [¹²⁵I]-ANP administration were detected, no or just few radioactivity was found after administration of [¹²⁵I]-C-ANP. On the other hand in the kidney papilla and the outer subcortical medulla, characteristic blackening was found after [¹²⁵I]-C-ANP administration. Those differences might be important for the understanding of pharmacological actions of ANP analogues.This work is part of the doctoral thesis of Frank Heidemann to be presented at the Ludwig-Maximilians-Universität München, FRG     

Surface receptors: Are they involved in transformation of spermatozoa of Ascaris?

Exposure of the spheroidal spermatozoa of Ascaris suum to an extract of the male accessory gland causes their transformation into ameboid cells. We have investigated the mechanism of this transformation, also termed activation, by labeling the proteins of accessory gland extracts with fluorescein isothiocyanate (FITC) or [125I], followed by qualitative localization of the sperm activating substances (SAS) and quantitative measurements of [125I]-SAS binding. Fluorescent patches of FITC-conjugated SAS were localized at the spermatozoan surface and were concentrated primarily at the posterior region. Few fluorescent patches were detectable in the region of the newly formed pseudopodia following transformation. Although spermatozoan transformation occurs within 2-5 min after exposure to SAS, the fluorescent patches became more distinct after a minimum of 8 min and reached maximum density at 15-30 min. Spermatozoa activated with [125I]-SAS became radioactively labeled in direct proportion to the amount of available [125I]-SAS until a saturation level was reached. SDS-polyacrylamide gel electrophoresis combined with autoradiography indicated that the cells bind two SAS components, of small (9,000 MW) and large (56,000 MW) sizes. These same two components were also detectable in a membrane fraction, obtained by differential centrifugation, of the spermatozoa after incubation with [125I]-SAS. binding of the two SAS components was not inhibited by preincubation of the spermatozoa with trypsin or Concanavalin A; however, the 56,000 MW component of SAS was not detectable in autoradiograms of spermatozoa incubated with periodic acid (1.6-10 mM) treated SAS. Such cells also failed to transform into ameboid spermatozoa. These results indicate that the two components of SAS that bind to the spermatozoan surface are possibly responsible for inducing the cell transformations associated with activation.     

Disposition of125I-labeled recombinant human tumor necrosis factor in the tumor-bearing mouse

Disposition of [¹²⁵I]rHu-TNF was elucidated in BALB/c mice bearing Meth A fibrosarcoma 7 days after transplantation. Afteri.v. administration, [¹²⁵I]rHu-TNF measured by radioactivity and immunoreactivity biphasically decreased in plasma. Tumor level of [¹²⁵I]rHu-TNF was the maximum at 1 h, then decreased and finally remained essentially constant. After i.t. administration, plasma level reached the maximum at 1 h. Tumor level decreased quickly and then became essentially constant. [¹²⁵I]rHu-TNF was suggested to be degraded to small fragments in the tumor. Significant distribution of [¹²⁵I]rHu-TNF was found in the kidney, lung, liver and tumor. Most tissue levels decreased with time in parallel with plasma levels. [¹²⁵I]rHu-TNF radioactivity was found in proximal convoluted tubules of kidney and in those areas of tumor consisting of degenerating cells with pyknotic nuclei. Urine contained most of administered radioactivity, which being neither immunoreactive nor protein-bound.     

Characterization of 5-HT transporter and receptor system in HeLaS3 cells by [H]8-OH-DPAT and other serotonergic ligands

[³H]8-OH-DPAT is a selective ligand for labeling 5-HT_1A receptor sites. In competition binding experiments, we found that classic biogenic amine transporter inhibitors displaced [³H]8-OH-DPAT binding at its high-affinity binding sites in HeLaS3 cells. [¹²⁵I]RTI-55 and [³H]paroxetine are known to specifically label amine transporter sites, and this was observed in our cells. Displacement studies showed that 8-OH-DPAT displayed affinity in a dose-dependent manner for the labeled amine transporter sites. These data suggest that [³H]8-OH-DPAT binds to amine uptake sites in HeLaS3 cells. A variety of drugs targeting different classes of receptors did not significantly affect [³H]8-OH-DPAT binding. Moreover, we determined the specific binding effects of various serotonergic ligands (i.e. [¹²⁵I]cyanopindolol, [³H]ketanserin/[³H]mesulergine, [³H]GR-65630, [³H]GR-113808 and [³H]LSD) that specifically labeled 5-HT₁, 5-HT₂, 5-HT₃, 5-HT₄ and 5-HT_5–7 receptors, respectively. It is suggested that HeLaS3 cells contain distinct types of the related to 5-HT receptor recognition binding sites. These observations could help elucidate the relevant characteristics of different types of 5-HT receptors and 5-HT membrane transporters in tumor cells and their role in tumorigenesis.     

Intracellular processing of residualizing labels in different cell types in vitro.

In previous autoradiographic studies on the sites of catabolism of rat serum albumin (RSA) in the rat, fibroblasts in skin and muscle were shown to accumulate degradation product from RSA labeled with the residualizing label dilactitol-125I-tyramine (125I-DLT) (Strobel et al., 1986 J. Biol. Chem., 261:7989-7994). Residualizing labels remain at the cellular site of degradation of the carrier protein because of their size, hydrophilicity, and resistance to lysosomal hydrolases. This study was designed to evaluate whether fibroblasts might retain labeled degradation products more efficiently than other cell types. The uptake of 125I-DLT-RSA and release of its degradation products and of a second non-biodegradable probe, fluorescein isothiocyanate (FITC)-dextran, were studied in fibroblasts, endothelial cells, and macrophages, all cell types previously implicated in the catabolism of albumin in vivo. The rates of uptake of labeled protein and dextran were comparable in all cell types and consistent with fluid phase endocytosis. The rate of release of both intact protein (30-35% of total radioactivity released) and radioactively labeled degradation products followed similar kinetics and had half-lives ranging from 26 to 37 hr. The rate of release of FITC-dextran was slower than that of radioactivity, with a half-life of 42-125 hr. Thus, although there were differences between the rates of release of the fluorescent and radioactive materials in vitro, there were no significant differences in the disposition of protein-derived catabolites among these three cell types.     

Determination of cell surface expression of Toll-like receptor 4 by cellular enzyme-linked immunosorbent assay and radiolabeling

Lipopolysaccharide (LPS) is recognized by Toll-like receptor 4 (TLR4) of macrophages triggering production of pro-inflammatory mediators. One of the factors determining the magnitude of responses to LPS, which may even lead to life-threatening septic shock, is the cell surface abundance of TLR4. However, quantitation of the surface TLR4 is difficult due to the low level of receptor expression. To develop a method of TLR4 assessment, we labeled the receptor on the cell surface with a rabbit antibody followed by either anti-rabbit immunoglobulin G–fluorescein isothiocyanate (IgG–FITC) for flow cytometry or with anti-rabbit IgG–peroxidase for a cellular enzyme-linked immunosorbent assay (ELISA). Alternatively, the anti-TLR4 antibody was detected by anti-rabbit IgG labeled with ¹²⁵I. Flow cytometry did not allow detection of TLR4 on the surface of J774 cells or human macrophages. In contrast, application of cellular ELISA or the radiolabeling technique combined with effective blockage of nonspecific binding of antibodies provided TLR4-specific signals. The level of TLR4 on the surface of J774 cells did not change on treatment with 1–100 ng/ml LPS; however, it was reduced by approximately 30–40% after 2 h of treatment with 1 μg/ml LPS. These data indicate that down-regulation of surface TLR4 can serve as a means of negative regulation of cell responses toward high doses of LPS.     

Complement protein C9 labeled with fluorescein isothiocyanate can be used to monitor C9 polymerization and formation of the cytolytic membrane lesion

Human complement protein C9 was covalently labeled with the fluorescent chromophore fluorescein isothiocyanate (FITC) with only a small reduction in the cytolytic activity of the protein. Polymerization of the labeled protein--either by incubating with lipid vesicles treated with complement proteins C5b-8 (activating the C5b-9 membrane lesion) or by heating the protein [Tschopp, J., Muller-Eberhard, H.J., & Podack, E.R. (1982) Nature (London) 298, 534]--resulted in a 40-60% decrease in the fluorescence emission from FITC. The decrease in total fluorescence was accompanied by an increase in the steady-state anisotropy following activation and polymerization of FITC-C9 by C5b-8 membranes, while heat-induced aggregation of the protein resulted in a dramatic depolarization of fluorescence. Only small changes in either the absorbance spectrum or fluorescence lifetime of the chromophore were detected upon FITC-C9 polymerization. Evidence is presented that the measured changes in FITC fluorescence upon C9 activation are due to self energy transfer between closely apposed fluorescein chromophores which occur in the polymerized form of the protein. The significance of these observations to the molecular structure of the assembled C5b-9 complex is discussed, as are the potential applications of this fluorescent derivative of C9.     

Effect of the label of oligosaccharide acceptors on the kinetic parameters of nasturtium seed xyloglucan endotransglycosylase (XET)

Fluorescently labeled derivatives of a xyloglucan (XG) nonasaccharide Glc₄Xyl₃Gal₂ (XLLG) were used as glycosyl acceptors in assays of xyloglucan endotransglycosylase (XET) from germinated nasturtium (Tropaeolum majus) seeds. We have investigated how the type of the oligosaccharide label influences the kinetic parameters of the reaction. The fluorescent probes used to label XLLG were anthranilic acid (AA), 8-aminonaphtalene-1,3,6-trisulfonic acid (ANTS), fluorescein isothiocyanate (FITC), and sulforhodamine (SR), respectively. The obtained data were compared with those of the reactions where aldose and/or alditol forms of tritium-labeled xyloglucan-derived nonasaccharide served as the respective acceptors. Modification at C-1 of the reducing-end glucose in XLLG by substitution with the fluorophore markedly affected the kinetic parameters of the reaction. The Michaelis constants K_m for individual acceptors increased in the order [1-³H]XLLG < XLLG-SR < [1-³H]XLLGol < XLLG-FITC < XLLG-ANTS < XLLG-AA, while the turnover numbers characterized by k_cat decreased in the order XLLG-FITC > XLLG-SR > XLLG-ANTS > [1-³H]XLLGol > [1-³H]XLLG > XLLG-AA. Catalytic efficiency (expressed as k_cat/K_m) with XLLG labeled with SR or FITC was 15 and 28 times, respectively, higher than with the tritium-labeled natural substrate [1-³H]XLLG. Comparison of the kinetic parameters found with acceptors labeled with different types of labels enables to select the most effective substrates for the high-throughput assays of XET.     

Presence of a common structure in the two molecular species of mouse L cell interferon

Tryptic digests of the two molecular species of purified mouse L cell interferon, labeled with [¹²⁵I] and [³H] methionine, were analyzed chromatographically. The 40,000 dalton-species yielded 4 methionine-containing and 6 [¹²⁵I]-labeled fragments, whereas the 24,000 dalton-species gave rise to 4 methionine- and 7 [¹²⁵I]-labeled fragments. Of these, 3 methionine-containing and 3 [¹²⁵I]-labeled fragments were found chromatographically identical between the two species. These results suggest that the two distinct species of interferon contain a common polypeptide structure.     

Some quantitative aspects of the labelling of proteins with 125I by the iodine monochloride method

The labelling of proteins by the iodine monochloride method was studied by using a mathematical model. The equations used were primarily derived from the mass law equation of the isotopic exchange reaction between [¹²⁵I]iodide and iodine monochloride. For convenient application, all equations were programmed into a computing desk-top calculator. To support the validity of the theoretical model, a series of iodinations of insulin were performed under various labelling conditions. The results of these experiments compare well with the theoretically derived values. Deviations from the theoretical values occurring at molar ratios of [¹²⁵I]iodide to iodine monochloride < 0.1 and > 4.0 are explained and suggestions made about how to prevent them. The mathematical model was used to simulate the isotopic exchange, and the iodination reaction under various conditions, to study (a) the influence of the amount of [¹²⁵I]iodide on the amount of [¹²⁵I]iodine monochloride formed, (b) the influence of the specific radioactivity of [¹²⁵I]iodide on the amount of [¹²⁵I]iodine monochloride formed, and (c) the influence of the specific radioactivity of [¹²⁵I]iodide on the number of millicuries needed for labelling to a desired extent.     

Affinity Labeling Mu Opioid Receptors With Novel Radioligands

1. A series of novel opiate ligands based upon 6α-naloxamine have been examined in opioid receptor binding assays.2. Coupling an ethylamine spacer alone to 6-α-naloxamine gave a compound with relatively poor affinity for mu opioid receptors compared to naloxone, although it retained high affinity for kappa₁ opioid receptors. Coupling a benzoyl group significantly increased the affinity. The presence at the 4-position of the benzoyl moiety of an amino-(NalAmiBen) or an azido-substituent (NalAziBen) did not significantly effect the affinity at mu receptors. However, iodinating the benzoyl moiety at the 3-position increased the affinity of the derivatives.3. Two compounds were radiolabeled and evaluated in receptor binding assays. Both radioligands labeled sites in CHO cells stably transfected with the mouse MOR-1 clone. The amino coupound [¹²⁵I]NalAmiBen and the azido derivative [¹²⁵I]NalAziBen reversibly bound to membranes from CHO cells transfected with MOR-1 with high affinity in the dark. Exposure of [¹²⁵I]NalAmiBen to UV did not alter the reversibility of binding, but exposure of [¹²⁵I]NalAziBen to UV light led to the covalent coupling of the radioligand to the receptor. When run on SDS-PAGE, [¹²⁵I]NalAziBen binding showed a band at approximately 70–80 kDa. A control corresponding to nonspecific binding failed to reveal any labeling. No bands were observed from membranes labeled with [¹²⁵I]NalAmiBen.     

Fluorescence labeling method for estimation of soluble polymers in the living material

The highly fluorescent derivatives of fluorescein, bearing the aliphatic primary amino groups, N-(2-aminoethylcarbonyl)-5(6)-aminofluorescein and 5-[N′-(2-aminoethyl)thioureido]fluorescein, were prepared for labeling of soluble polymers. The absorption and emission properties of these labels and polymers labeled with them were compared with properties of fluorescein and fluorescein isothiocyanate (FITC)-bovine serum albumin conjugate. Effects of the chemical structure of the polymer on the relative fluorescence quantum yield of a covalently attached label were evaluated using ionogenic, olefinic, or phenolic groups in side chains. The fluorescence of labeled polymers was adequate for their tracing in all the cases studied. The most pronounced quenching of fluorescence in the presence of phenolic groups is comparable with the quenching of fluorescence of FITC observed in FITC-protein conjugates. The long-term stability of the polymer-fluorochrome bond was checked in solutions of pH 2.10, 7.46, and 11.84; a higher stability of simple amide over amide plus the thiourea bond was found. The quantitative method of measurement of the concentration of labeled polymers in the biological material in a range of about 10 ng was developed; factors affecting the reproducibility are discussed.