Magnetic sorting of leukocytes

Magnetic filtration of surface-labeled cells has been applied to the fractionation of leukocytes in a model system, using a colloidal magnetite reagent to label mouse spleen cells. This reagent was completely free from problems of aggregation or settling. Since the individual submicron particles were invisible under the microscope, cells were not visibly altered by labeling. Viability also was unaffected by either labeling or magnetic filtration. Using a 10-kG magnet and a 5-ml filter column, 50 million cells were fractionated in less than 10 min, with 99% removal of labeled T lymphocytes. The efficiency of the magnetic method is limited at present by the fact that cells that do not have the surface target antigen of interest, and so are not antibody coated, may adsorb a small amount of label nonspecifically. These then have a nonzero chance of being captured in the filter along with the labeled cells.     

Separation of cells labeled with immunospecific iron dextran microspheres using high gradient magnetic chromatography

Immunospecific magnetic microspheres, consisting of ferromagnetic iron dextran conjugated to Protein A, were used to specifically label red blood cells (RBC) for cell separation studies using high gradient magnetic chromatography ( HGMC ). When 10(7)-10(8) RBC labeled with Protein A-iron dextran microspheres were applied to a column containing 30 mg stainless steel wire placed in a 7.5 kilogauss magnetic field, 96 +/- 2% of the cells were retained in the column. These cells could be eluted by removing the magnetic field and mechanically agitating the column. The retention of labeled cells by HGMC was shown to be dependent on the applied magnetic field and the amount of wire packed into the column. HGMC in conjunction with cell labeling with immunospecific iron dextran microspheres have useful applications for the separation of specific cell types.     

Quantitative Proteomics Employing Primary Amine Affinity Tags

A proteomics-based method using stable isotope labeling to assess the relative abundance of peptides or proteins is described. Bradykinin and carbonic anhydrase were labeled with sulfosuccinimidyl-2-(biotinamido) ethyl-1,3-dithiopropionate, a membrane impermeant reagent that is reactive with primary amines. Specificity of the label to primary amines was demonstrated using tandem mass spectrometry. Also, relative quantitation was achieved by secondary labeling with natural isotopic abundance and stable isotope-labeled methyl iodide. We believe this to be an effective stable isotope-labeling method for quantitative proteomics.     

Photoaffinity labeling of the insulin receptor in H4 hepatoma cells: Lack of cellular receptor processing

Photoaffinity labeling techniques were used to identify insulin-binding components of the plasma membrane in insulin-responsive, monolayer-cultured hepatoma cells. The activated, photosensitive reagent, an n-hydroxysuccinimide ester of 4-azidobenzoic acid, was coupled with highly purifed insulin, and the hormone derivative was subsequently iodinated, bound to cell surface receptors of intact H4 cells, and photoactivatcd. After dissolution of the cells, labeled proteins were analyzed by SDS/polyacrylamide gel electrophoresis under reducing conditions. The main labeled band exhibited an apparent molecular weight of 130,000. Two minor components of apparent mol wt 95,000 and 40,000 were also identified. Specific labeling of all 3 bands was inhibited by simultaneous incubation of the cells with native insulin, but not by the heterologous hormone, glucagon, prior to photoactivation. Binding of azidobenzoyl-insulin to H4 cells was time-dependent, as was the correlated labeling of receptor components. Band-labeling by the photosensitive insulin derivative was totally light-dependent; spontaneous covalent linking of insulin and receptor was not observed. The labeled receptor-related proteins were not degraded by the cells under our experimental conditions.     

Theoretical study to gain fundamental insight into reaction mechanism of N–S acyl transfer of N-sulfanylethylanilide-based protein labeling reagent on protein surface

Elucidation of protein function is one of the central issues in the field of life sciences. To study the function of proteins not in isolation, but in a cell or its lysate, thus, it is necessary to selectively label the target protein in a mixture. Affinity labeling is one of several widely used methods for selective labeling; however, this method has the disadvantage that the labeling reagent is always activated, albeit weakly. Therefore, fine-tuning of the reactivity and/or reaction conditions is generally required for successful target-selective labeling. We previously developed a new affinity labeling reagent with N-sulfanylethylanilide (SEAlide) as a key reactive unit. It was designed based on the following hypotheses. SEAlide is less reactive and does not label in the absence of a target protein. Upon target binding, amino acid side-chain functional groups on the target surface convert SEAlide into a thioester form via N–S acyl transfer, allowing the target to be labeled. However, no evidence has been obtained so far to directly prove the hypothesis. In this study, we examine whether amino acid side-chain functional groups can activate SEAlide from the viewpoint of theoretical chemistry. The theoretical studies show that the activation free energy and enthalpy of the acyl transfer of SEAlide are reduced in the presence of methylammonium, which is a model for the protonated side chain of Lys, and acetate, which is a model for the deprotonated side chain of Asp/Glu. It suggests that Lys and Asp/Glu side chains could potentially stabilize the activation transition states to accelerate the thioester formation. Furthermore, the significant decrease in the activation enthalpy indicates that the contribution of entropy to the transition state is large. This result supports the original hypothesis that the SEAlide-based labeling reagent is efficiently activated by binding to the target protein.     

Radio-iodination of plasma membranes of toad bladder epithelium

Summary The present report describes high yield enzymatic radio-iodination of the apical and basal-lateral plasma membranes of toad bladder epithelium, by a procedure that does not breach the functional integrity of the epithelium, as assessed by the basal and vasopressin-sensitive short-circuit current (SCC). Restriction of the label to the membrane surface was ascertained by light and electron-microscopic autoradiographs. On the apical surface, the grains were over the glycocalyx and the plasma membrane. Analysis of the labeled glycocalyx by agarose gel filtration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), as well as enzymatic and pH-dependent hydrolysis indicated that the glycocalyx is a trichloro-acetic acid-soluble macromolecular complex of high molecular weight composed of a peptide moiety attached to large prosthetic groups (presumably carbohydrates) by O-glycosidic bonds. Analysis of the labeled apical plasma membrane components by agarose gel filtration and SDS-PAGE disclosed the presence of six major species of apparent molecular weights: 23,000, 28,000, 37,000, 44,000, 68,000, and 95,000. More than half of the membrane-associated radio-iodine was in two bands of molecular weights 37,000 and 44,000.Concentrations of vasopressin and cyclic AMP sufficient to increase the SCC significantly did not modify the extent of membrane labeling or the distribution of the label among the apical membrane components (presumably proteins) as assessed by SDS-PAGE. Iodination in the presence of amiloride inhibited incorporation but did not change the pattern of the distribution of the label among the components resolved by SDS-PAGE.Iodination of basal-lateral plasma membranes, at a yield comparable to that obtained with apical labeling, was attained after about 30 min of exposure of the intact bladder to the labeling solutions. Approximately 25% of the basal-lateral labeling was lost when the epithelial cells were harvested after collagenase treatment, implying that some iodination of the basement membrane had taken place. Less than 10% of iodination of the apical or basal-lateral surfaces was accounted for by lipid-labeling. Analysis of the labeled apical and basal-lateral species by enzymatic digestion and thin layer chromatography disclosed that virtually all the radioactivity was present as mono-iodotyrosine (MIT).     

Labeling and chemistry of grapefruit pectic substances

The labeling of a number of polysaccharides found in grapefruit (Citrus paradisi) was achieved by feeding labeled myo-inositol to ripening grapefruit through their cut fruit stem, and allowing 4 days for the metabolism of label. The pectic polysaccharides were isolated by successive extraction of the labeled grapefruit with 80% ethanol, chloroform-methanol (1:1) and finally with 0.2 M Na₂ EDTA to solubilize pectic polysaccharides. The incorporation of label from myo-inositol into galacturonosyl, arabinosyl, xylosyl and galactosyl residues of pectic polysaccharides via myo-inositol oxidation pathway was demonstrated. Ion exchange chromatography of these labeled pectic polysaccharides using DE-52 cellulose resulted in the elution of eight totally or partially resolved polysaccharides with increasing salt concentration. The results suggest that, like other plant tissues, the myo-inositol oxidation pathway is also operative in ripening grapefruit and this metabolic pathway could be successfully utilized to achieve labeling of a number of pectic polysaccharides.     

Optimization of 5-(and-6)-Carboxyfluorescein Diacetate Succinimidyl Ester for Labeling Human Intervertebral Disc Cells in Vitro

We have assessed the utility of an intracellular fluorochrome, 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE), as a tracking label for human intervertebral disc cells in vitro. Although 5 JJIM provides adequate intracellular labeling for whole cell fluorescent microscopic identification of labeled cells, 20 JJLM was preferable for immunocytochemical localization of paraffin embedded labeled cells. Electron dense vesicles are seen at the ultra-structural level in labeled cells. Discrete vesicular labeling can also be observed in whole cell mounts viewed with fluorescence microscopy. Whole cells retain good label for 6 weeks. CFSE labeling is relatively easy, nontoxic to cells and nonradiocactive. Initial optimization of dose with specific cells types is recommended when confirmation of positive immunocytochemistry is needed for tissue engineering studies.     

Changes in retention behavior of fluorescently labeled proteins during ion-exchange chromatography caused by different protein surface labeling positions

Confocal laser scanning microscopy (CLSM) is a method allowing in situ visualization of protein transport in porous chromatography resins. CLSM requires labeling a protein with a fluorescent probe. Recent work has shown that conjugation of the protein with fluorescent probes can lead to significant changes in the retention time of the protein-dye conjugate with respect to the unlabeled protein. In this study, we show that common labeling procedures result in a heterogeneous mixture of different variants and that attachment location of the fluorescent probe on the protein surface can have a strong effect on the retention of protein-dye conjugate. Lysozyme was labeled with Cy5 and BODIPY-FL succinimidyl esters, followed by chromatographic separation of the different lysozyme-dye conjugates and subsequent determination of the label position using MALDI-TOF-MS. Finally, homogenously labeled lysozyme-dye conjugates were used in CLSM experimentation and compared to published results arising from heterogeneously labeled feedstocks. The results confirm that the attachment location of the fluorescent probe has a strong effect on chromatographic retention behavior. When addressing the binding affinities of the different labeled protein fractions, it was found that native lysozyme was able to displace lysozyme-dye conjugates when the fluorescent label was attached to lysine-33, but not when attached to lysine-97. Finally, it could be shown that when superimposing the single profiles of the three major fractions obtained during a labeling procedure a qualitative picture of the net profile is obtained.     

Efficient labeling of mesenchymal stem cells using cell permeable magnetic nanoparticles

For the purpose of successfully monitoring labeled cells, optimum labeling efficiency without any side effect is a prerequisite. Magnetic cellular imaging is a new and growing field that allows the visualization of implanted cells in vivo. Herein, superparamagnetic iron oxide (SPIO) nanoparticles were conjugated with a non-toxic protein transduction domain (PTD), identified by the authors and termed low molecular weight protamine (LMWP), to generate efficient and non-toxic cell labeling tools. The cells labeled with LMWP-SPIO presented the highest iron content compared to those labeled with naked SPIO and the complex of SPIO with poly-l-lysine, which is currently used as a transfection agent. In addition to the iron content assay, Prussian staining and confocal observation demonstrated the highest intracellular LMWP-SPIO presence, and the labeling procedure did not alter the cell differentiation capacity of mesenchymal stem cells. Taken together, cell permeable magnetic nanoparticles conjugated with LMWP can be suggested as labeling tools for efficient magnetic imaging of transplanted cells.     

Optimization of 5-(and-6)-Carboxyfluorescein Diacetate Succinimidyl Ester for Labeling Human Intervertebral Disc Cells in Vitro

We have assessed the utility of an intracellular fluorochrome, 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE), as a tracking label for human intervertebral disc cells in vitro. Although 5 JJIM provides adequate intracellular labeling for whole cell fluorescent microscopic identification of labeled cells, 20 JJLM was preferable for immunocytochemical localization of paraffin embedded labeled cells. Electron dense vesicles are seen at the ultra-structural level in labeled cells. Discrete vesicular labeling can also be observed in whole cell mounts viewed with fluorescence microscopy. Whole cells retain good label for 6 weeks. CFSE labeling is relatively easy, nontoxic to cells and nonradiocactive. Initial optimization of dose with specific cells types is recommended when confirmation of positive immunocytochemistry is needed for tissue engineering studies.     

Chemical modification and labeling of glutamate residues at the stilbenedisulfonate site of human red blood cell band 3 protein

A new method has been developed for the chemical modification and labeling of carboxyl groups in proteins. Carboxyl groups are activated with Woodward's reagent K (N-ethyl-5-phenylisoxazolium 3'-sulfonate), and the adducts are reduced with [3H]BH4. The method has been applied to the anion transport protein of the human red blood cell (band 3). Woodward's reagent K is a reasonably potent inhibitor of band 3-mediated anion transport; a 5-min exposure of intact cells to 2 mM reagent at pH 6.5 produces 80% inhibition of transport. The inhibition is a consequence of modification of residues that can be protected by 4,4'-dinitrostilbene-2,2'-disulfonate. Treatment of intact cells with Woodward's reagent K followed by B3H4 causes extensive labeling of band 3, with minimal labeling of intracellular proteins such as spectrin. Proteolytic digestion of the labeled protein reveals that both the 60- and the 35-kDa chymotryptic fragments are labeled and that the labeling of each is inhibitable by stilbenedisulfonate. If the reduction is performed at neutral pH the major labeled product is the primary alcohol corresponding to the original carboxylic acid. Liquid chromatography of acid hydrolysates of labeled affinity-purified band 3 shows that glutamate but not aspartate residues have been converted into the hydroxyl derivative. This is the first demonstration of the conversion of a glutamate carboxyl group to an alcohol in a protein. The labeling experiments reveal that there are two glutamate residues that are sufficiently close to the stilbenedisulfonate site for their labeling to be blocked by 4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate and 4,4'-dinitrostilbene-2,2'-disulfonate.     

红外荧光素Alexa Fluor 647标记的2型腺相关病毒保持天然的感染活性

近年来红外荧光素的研发及相应的标记技术的进展在生物医学领域的研究中备受关注,通过与尖端仪器的结合在一系列生物样本的检测中得到广泛的应用. 然而,红外荧光标记技术在病毒感染生物学研究中的应用却少见报道.本文建立了Alexa Fluor 647（AF647）红外荧光素标记重组2型腺相关病毒（AAV2）的方法,并通过凝胶电泳的银染、红外荧光成像和透射电镜分析等手段,对标记病毒的结构和形态与未标记的病毒进行了细致的比较,同时进一步鉴定了其感染293T细胞后的转导活性.结果发现,AAV2在标记后的理化性质和感染活性均未发生改变. 应用狭缝印迹和激光共聚焦显微成像技术,分别对标记病毒的细胞摄取效率和在宿主细胞内的分布进行描述,为红外标记AAV2在研究中的应用提供了范例.AF647标记的 AAV2为腺相关病毒的感染动力学研究提供了新的手段,具有敏感度高、抗荧光漂白和低非特异干扰等优点,并且对直接有效地结合形态学分析和生化分子生物学检测,具有重要的应用价值.     

End-labeling of long DNA fragments with biotin and detection of DNA immobilized on magnetic beads

To immobilize DNA fragments onto magnetic beads coated with streptavidin for isolation purpose, it is important to label one biotin molecule at one terminus of DNA fragment. After failure to label long DNA with biotin by PCR and filling-in reaction, a 9.2 kb DNA was labeled with biotin by a modified ligation strategy. A simple method is also reported to detect the quantity and integrity of DNA immobilized on the magnetic beads.     

Labeling of proteins with [35S]methionine and/or [35S]cysteine in the absence of cells

Incubation of [35S]methionine and [35S]cysteine with bovine albumin, globulin, catalase, hemoglobin, or human globulin resulted in incorporation of the 35S label into each of these proteins. Trichloroacetic acid (TCA) precipitation revealed that the percentage of label incorporated ranged from 1 to 15%. The 35S labeling was resistant to dissociation by reducing SDS-PAGE, prolonged dialysis against 4 M urea, heating, TCA precipitation, and dilution by gel filtration. The labeling effect was more efficient with [35S]cysteine than [35S]methionine. Incubation of 35S label with proteins differing in methionine and cysteine content revealed no requirement for sulfur-containing amino acids in the target protein. Protein carboxymethylation reduced but did not prevent 35S label incorporation. Amino acid analysis of labeled proteins revealed that the radioactive label was not consistently associated with an individual amino acid. Differences in the ability of various proteins to spontaneously label with these amino acids suggest caution in the interpretation of metabolic labeling experiments and the necessity for inclusion of additional controls. Alternatively, our experience indicates a potentially useful method for labeling proteins in the absence of cells.     

Platform development for expression and purification of stable isotope labeled monoclonal antibodies in Escherichia coli

The widespread use of monoclonal antibodies (mAbs) as a platform for therapeutic drug development in the pharmaceutical industry has led to an increased interest in robust experimental approaches for assessment of mAb structure, stability and dynamics. The ability to enrich proteins with stable isotopes is a prerequisite for the in-depth application of many structural and biophysical methods, including nuclear magnetic resonance (NMR), small angle neutron scattering, neutron reflectometry, and quantitative mass spectrometry. While mAbs can typically be produced with very high yields using mammalian cell expression, stable isotope labeling using cell culture is expensive and often impractical. The most common and cost-efficient approach to label proteins is to express proteins in Escherichia coli grown in minimal media; however, such methods for mAbs have not been reported to date. Here we present, for the first time, the expression and purification of a stable isotope labeled mAb from a genetically engineered E. coli strain capable of forming disulfide bonds in its cytoplasm. It is shown using two-dimensional NMR spectral fingerprinting that the unlabeled mAb and the mAb singly or triply labeled with ¹³C, ¹⁵N, ²H are well folded, with only minor structural differences relative to the mammalian cell-produced mAb that are attributed to the lack of glycosylation in the Fc domain. This advancement of an E. coli-based mAb expression platform will facilitate the production of mAbs for in-depth structural characterization, including the high resolution investigation of mechanisms of action.     

Affinity labeling of the 5-methyltetrahydrofolate/methotrexate transport protein of L1210 cells by treatment with an N-hydroxysuccinimide ester of [3H]methotrexate

An N-hydroxysuccinimide ester of [3H]methotrexate has been employed to covalently label a specific binding protein that resides in the plasma membrane of L1210 cells. Incorporation of radioactivity into this protein accounted for 55% of total cellular labeling, was half-maximal at a reagent concentration of 27 nM, and was blocked either by prior exposure to unlabeled reagent or by the addition of excess methotrexate. A role for this protein in methotrexate transport was supported by the observations that: (a) similar concentrations of reagent were required for both labeling of the binding protein and irreversible inhibition of transport; (b) the amount of labeled binding protein was comparable to observed levels of transport protein; (c) protection against labeling was afforded by thiamin pyrophosphate, a potent competitive inhibitor of methotrexate transport; and (d) labeling of the binding protein was not observed in a subline of L1210 cells that has a defect in the ability to transport methotrexate. The binding protein could be solubilized from the membrane by various ionic and non-ionic detergents and the covalent bond between the incorporated [3H]methotrexate and the protein was stable to a variety of conditions, including high concentrations of mercaptoethanol and hydroxylamine and extremes of pH. The labeled protein fractionated as a nearly symmetrical peak on Sephacryl S-300 and it appeared as a single band (Mr = 36,000) after electrophoresis in polyacrylamide gel containing sodium dodecyl sulfate.     

Characterization of the Pre-meiotic S Phase through Incorporation of BrdU during Spermatogenesis in the Rat

Seminiferous tubules in mammals have histological arrangements defined by the associations between somatic cells and germ cells. The processes of DNA synthesis in meiotic and mitotic cells have different features that are not easily distinguishable through morphological means. In order to characterize the pre-meiotic S phase, 5-bromo-2’-deoxyuridine (BrdU) was injected intraperitoneally into Wistar rats, which were sacrificed 30 min, 2 hr, and 24 hr after injection. We found three different labeling patterns. One of these patterns was characterized by a distribution of the label in the form of speckles, most of which were associated with the nuclear envelope (labeling type I). We suggest that this pattern is due to mitotic DNA synthesis of type B spermatogonia. Labeling type II consisted of labeled foci scattered throughout the nuclear volume, which can be correlated with preleptotenic cells in pre-meiotic DNA synthesis. After 24 hr of incorporation, a third type of labeling, characterized by large speckles, was found to be related to cells in the “bouquet” stage; that is, cells in transition between the leptotene and zygotene phases. Our results indicate that BrdU incorporation induces different labeling patterns in the mitotic and pre-meiotic S phases and thus makes it possible to identify somatic and germinal cells.     

Sequential expression of cell surface C3bi receptors during neutrophil locomotion

Fluorescence microscopy has been used to study the cell surface distribution of the complement receptor for C3bi (CR3) on human neutrophils during locomotion. CR3 is an integral membrane protein that participates in cell attachment phenomena including chemotaxis. Fluorescein- and rhodamine-conjugated monoclonal IgG or Fab fragments were used to label CR3. We have previously shown that CR3 is uniformly distributed on unstimulated cells. During cell locomotion the fluorescent labels redistribute to the uropod and retraction fibers. To better understand the role of CR3 in chemotaxis, we have performed sequential two-color labeling experiments in conjunction with fluorescence microscopy. Double-labeling experiments were conducted by labeling adherent neutrophils with fluorescein-conjugated anti-CR3 followed by chemotaxis in a gradient of FMLP (10(-7) M). The cells were then labeled again with rhodamine-conjugated anti-CR3. The uropod and distal training filopodia were labeled with fluorescein, whereas the cell body and occasionally proximal filopodia near the uropod were labeled with rhodamine. When neutrophils were fixed and permeabilized prior to the second CR3 labeling, the second fluorescent label was localized to a granule-like compartment(s), often near the lamellipodium. The results suggest a flow of CR3 from intracellular granules----lamellipodia and cell body----uropod----trailing filopodia during chemotaxis.     

Rapid mass spectrometric analysis of 15N-Leu incorporation fidelity during preparation of specifically labeled NMR samples

Advances in NMR spectroscopy have enabled the study of larger proteins that typically have significant overlap in their spectra. Specific (15)N-amino acid incorporation is a powerful tool for reducing spectral overlap and attaining reliable sequential assignments. However, scrambling of the label during protein expression is a common problem. We describe a rapid method to evaluate the fidelity of specific (15)N-amino acid incorporation. The selectively labeled protein is proteolyzed, and the resulting peptides are analyzed using MALDI mass spectrometry. The (15)N incorporation is determined by analyzing the isotopic abundance of the peptides in the mass spectra using the program DEX. This analysis determined that expression with a 10-fold excess of unlabeled amino acids relative to the (15)N-amino acid prevents the scrambling of the (15)N label that is observed when equimolar amounts are used. MALDI TOF-TOF MS/MS data provide additional information that shows where the "extra" (15)N labels are incorporated, which can be useful in confirming ambiguous assignments. The described procedure provides a rapid technique to monitor the fidelity of selective labeling that does not require a lot of protein. These advantages make it an ideal way of determining optimal expression conditions for selectively labeled NMR samples.