Flow cytometry is commonly used to characterize cellular antigens expressed both on the cell surface and inside the cell. As the technology advances, more researchers are using this tool to study intracellular activity to understand how cells communicate and respond to their environment. Intracellular targets of interest include cytoplasmic antigens and proteins, nuclear antigens and proteins, cytokines, etc. The quality of intracellular staining depends on the ability of the permeabilizing reagent to permeabilize the cell membrane for antibodies to get to the target of interest without destroying the cell. Five different commercial permeabilizing reagents were evaluated in a small study, Caltag Fix and Perm® Kit, BD IntraSure™ Kit**This product is produced under license from Leeds Teaching Hospitals NHS Trust., BD FACS™ Permeabilizing Solution 2 (FACS Perm 2), 1X FACS Lysing Solution, and BD Cytofix/Cytoperm™ Kit. Five categories of performance were evaluated: cell recovery, maintenance of surface staining, preservation of light scattering properties, intracellular staining, and background staining. Cell recovery was assessed using CD45 APC and BD TruCOUNT™ tube; surface staining maintenance was assessed using CD45 APC, CD45 PerCP-Cy5.5, CD19 PE, and CD20 PerCP; intracellular and background staining were assessed using anti-bcl-2 FITC, anti-Zap 70 PE, anti-TdT FITC, anti-Ki-67 FITC, and CD3 FITC/anti-MPO PE/CD79a PerCP-Cy5.5 reagents. Caltag and BD IntraSure Kits performed well in the category of cell recovery. Caltag Kit, BD IntraSure Kit, and FACS Lysing Solution maintained cell surface staining after cell permeabilization. BD IntraSure Kit and FACS Lysing Solution preserved light scattering properties of cells after permeabilization. Using signal-to-noise ratio as the criterion to evaluate background and intracellular positive staining, FACS Lysing Solution and FACS Perm 2 detected anti-bcl-2 FITC; BD IntraSure Kit and FACS Lysing Solution detected anti-TdT FITC; BD IntraSure Kit, FACS Lysing Solution, FACS Perm 2, and BD Cytofix/Cytoperm Kit detected anti-Ki-67 FITC; BD IntraSure Kit, FACS Lysing Solution, and FACS Perm 2 detected anti-Zap 70 PE; all kits detected CD3 FITC/anti-MPO PE/CD79a PerCP-Cy5.5. Results showed that there is no universal permeabilizing reagent for all intracellular staining assays. Each test reagent has its own strength and weakness. Choosing the best permeabilizing reagent for a particular assay depends on the target of interest and the output of the assay.

Summary.CD38 expression on chronic lymphocytic leukaemia (CLL) cells is a poor prognostic factor, however, methods for measuring this vary. The QuantiBRITETMflow cytometry (FC) system yields an absolute antigen expression value (antibodies bound/cell, ABC) and may be useful in standardizing CD38 expression analysis. We evaluated cryopreserved pretreatment CLL cells for CD20 ABC, CD38 ABC, and percentage of CD38+B cells from 131 patients requiring therapy. The 92 patients (70%) with ≥ 100 CD38 ABC had worse overall survival (OS; 34% at 5 years) compared with those with < 100 CD38 ABC (70% at 5 years, mortality hazard ratio 2·30, 95% confidence interval 1·28–4·12; two‐tailedP = 0·003). Among the 64 patients with < 30% CD38+cells, OS of the 25 with ≥ 100 ABC was worse than that of the 39 with < 100 ABC (P = 0·018). OS of patients with < 30% CD38+cells and ≥ 100 ABC was actually similar to that of patients with ≥ 30% CD38+cells.BrightCD20 expression (≥ 20 000 ABC) was not associated with a worse OS (P = 0·10). The presence of ≥ 100 CD38 ABC in CLL patients requiring therapy is an unfavourable prognostic factor for OS and quantitative FC may be superior to percentage CD38+cell assessment. Prospective trials are required to determine more precisely the prognostic significance of absolute expression levels in fresh CLL cells.

An unusual population of high side scatter, low nucleic acid dye binding, dim CD45 cells was found in aged blood samples stained with the ProCOUNT reagent. Cell surface staining showed that these cells have the surface phenotype of neutrophils. However, they have decreased expression of several surface antigens, bind annexin V, and stain more dimly than normal neutrophils with LDS-751. These characteristics indicate that the cells have become apoptotic. The decreased expression of the CD45 antigen on apoptotic neutrophils could have an impact on some methods for enumerating CD34-positive progenitor cells. Absolute CD34-positive cell counts are frequently obtained by multiplying CD34-positive cells as a percentage of the total cells by the white blood cell count from a hematologyanalyzer. Cells staining dimly with CD45 may not pass a flow cytometer threshold set on this parameter but appear to be detected by a hematology instrument thresholding on cell size. Thus the white blood cell counts from the two sources may not be identical, introducing error into the calculated absolute CD34 count. Absolute counts of CD34 cells based on simultaneous acquisition of a counting bead are not affected by the presence of this population, but purity estimates can be affected if the possible presence of these cells is not considered in aged samples.

In the present study, we investigated whether apoptosis in hematopoietic cells is associated with changes in cellular phosphotyrosine content. Murine thymocytes and B cells, human leukemia cells, and normal peripheral blood leukocytes were induced to undergo apoptosis by treatment with specific stimuli or by incubation in growth factor-deprived medium. Multiparameter flow cytometry was used to measure changes in phosphotyrosine content that correlated with the appearance of features of programmed cell death, such as cell shrinkage, DNA fragmentation, and loss of membrane integrity. The results show that conditions that induced apoptosis also induced a dramatic decrease in cellular phosphotyrosine levels. Tyrosine dephosphorylation preceded the loss of plasma membrane integrity and, in most cases, was temporally correlated with the onset of DNA fragmentation. The protein tyrosine phosphatase antagonist pervanadate had a dose-dependent inhibitory effect on both dephosphorylation and apoptosis in murine thymocytes, which were treated with dexamethasone or with the topoisomerase II inhibitor etoposide. The results suggest that extensive tyrosine dephosphorylation is an intrinsic part of the apoptotic process of hematopoietic cells and may be involved mechanistically in the apoptosis induced by certain stimuli.

In an effort to improve detection of P-glycoprotein-mediated multidrug resistance (mdr), several dyes were compared to rhodamine 123 (R123) in efflux assays. Two dyes (SY-38 and SY-3150) were found that provided better sensitivity. These dyes were effluxed by a cell line known to be mdr-positive (P388/R84) but not by an mdr-negative cell line (P388). Efflux was blocked by both verapamil and cyclosporine A. Efflux from KG1a cells was less than from P388/R84, just as has been seen with R123 and daunomycin. In further experiments, a model system was used to demonstrate two-color immunofluorescence plus efflux measurements. This was done using a sequential staining method. A procedure was devised that, at least for this model system, allowed single-step staining with both dye and antibody. The sensitivity of the efflux measurement was slightly compromised by using this one-step staining method.

Apoptotic thymocytes were found to be much dimmer than normal thymocytes when stained with several nucleic acid dyes. These dyes provide a quick and simple assay for apoptosis which works for live cells and does not require a UV laser. The collection of dyes giving this staining pattern includes reagents suitable for use in either the FL1, FL2, or FL3 channel of a standard FACScan. Cells identified by these reagents were identical to apoptotic thymocytes defined by several widely used criteria: (i) rapid uptake of Hoechst 33342 but exclusion of propidium iodide, (ii) merocyanin 540 bright, and (iii) sub-G1 DNA content when permeabilized in a buffer that elutes fragmented DNA. In addition, L3T4/Thy-1 dim thymocytes were included in the dyc dim population. The standard Hoechst 33342 and merocyanin 540 assays were not able to separate the normal and apoptotic populations in HL-60 cells treated with camptothecin. However, the dyes SYTO-16 and LDS-751 both gave adequate differentiation of apoptotic from nonapoptotic cells in this model system. Some of these dyes also emit very little in other fluorescence channels of the flow cytometer and can be used in multicolor assays on cytometers equipped with only a single argon-ion laser.

An experimental flow cytometer was constructed using a quartz flow cell optically coupled to a 1.22 NA lens. A pair of crossed cylindrical quartz lenses allowed multilaser excitation. Two helium-cadmium (HeCd) lasers, emitting 16 mW at 442 nm and 35 mW at 325 nm, were used to excite chromomycin A3 and Hoechst 33258 fluorescence, respectively. Bivariate flow karyotypes from normal human male chromosomes and from the Daudi cell line were obtained and were compared to those from a standard instrument using dual water-cooled lasers. The new experimental instrument exhibited comparable resolution to that from the standard instrument. In further experiments with Daudi chromosomes, the 35 mW HeCd laser was replaced with a 10 mW HeCd laser, and the system still gave good, though slightly decreased, resolution.

A method was developed that uses paired DNA dyes to detect the incorporation of bromodeoxyuridine (BrdUrd) into cellular DNA and requires only 488 nm excitation. The fluorescence of thiazole blue, TO-PRO-3, and LDS-751 was found to be enhanced by the presence of BrdUrd in DNA. Pairing LDS-751, thiazole blue, or TO-PRO-3 with propidium iodide (PI) for flow cytometry allowed the differentiation of cells containing BrdUrd from BrdUrd unlabeled cells. LDS-751 can be excited directly at 488 nm, and TO-PRO-3 or thiazole blue can be excited indirectly by resonance energy transfer from PI. The enhancement of fluorescence from these dyes is correlated with a decrease in PI fluorescence, suggesting an increased energy transfer from PI to the red emitting dye. Fluorescence from other dyes, including thiazole orange, TO-PRO-1, rhodamine 800, oxazine 750, and 7-aminoactinomycin D, was not altered by the presence of BrdUrd in DNA. Results also were obtained showing that BrdUrd detection using PI and TO-PRO-3 is compatible with immunofluorescence staining with FITC-labeled antibodies.

Two Fab fragments of the monoclonal anti dinitrophenyl (DNP) spin-label antibody AN02 were prepared by recombination of specifically deuterated heavy and light chains. In the recombinant H(I)L(II) all the tyrosines and phenylalanines were perdeuterated as were the tryptophan residues of the heavy chain. In the recombinant H(II)L(I) all the tyrosines and phenylalanines were perdeuterated as were the tryptophan residues of the light chain. Saturation of three resonances of H(I)L(II), assigned to tryptophan protons of the light chain, resulted in magnetization transfer to the aromatic proton at position 6 of the DNP ring and to the CH2 protons of the glycines linked to the DNP in a diamagnetic hapten (DNP-DG). Saturation of three resonances of H(II)L(I) assigned to tryptophan protons of the heavy chain resulted in magnetization transfer to the CH2 protons of the glycines in DNP-DG. From the dependence of the magnetization transfer on the irradiation time, the cross relaxation rates between the involved protons were estimated. The inferred distances between these protons of the hapten and certain tryptophan protons are 3-4 A. It is concluded that in the combining site of AN02 there is one tryptophan from the light chain and one tryptophan from the heavy chain that are very near the hapten. When all tyrosines and phenylalanines were perdeuterated and all tryptophan aromatic protons were deuterated except for the protons at positions 2 and 5, titration of the Fab fragments with variable amounts of paramagnetic hapten showed that one proton from the light chain tryptophan is near (less than 7 A) the unpaired electron and that three other protons are significantly closer than 15 A.(ABSTRACT TRUNCATED AT 250 WORDS)

Nuclear magnetic resonance (NMR) has been used extensively to study the structure of antibody combining sites. In recent studies we have observed the proton resonance spectra of the Fab fragment of a monoclonal anti-spin-label antibody derived from a hybridoma grown on various specifically deuterated amino acids. The broadening of the proton resonance signals by the paramagnetic hapten, together with selective deuteration, has allowed the identification of most of the amino acids in the combining-site region of this antibody and has also provided estimates of distances between amino-acid protons and the unpaired electron. Here we show how recombination of specifically deuterated heavy and light chains permits the assignment of single amino-acid proton resonance signals to either the heavy or light chain. In addition, the spectra of such recombinants demonstrate that their combining-site structures must be almost identical to the native structure.

The nuclear magnetic resonance spectra of monoclonal Fab antibody fragments have been recorded in the absence and presence of the specific spin-label dinitrophenyl hapten. The difference spectra reveal the presence of about 50 amino acids in the region of the combining site. By selective deuteration and by use of double difference spectra, all the resonances in the spectral region -1 to 1.5 ppm have been identified. We have found that in the combining site region there are four or five valines, certainly three and possibly five threonines, three or four leucines, two or three isoleucines, and six or seven alanines. Selective deuteration of methionine and lysine reveals one methionine and two lysines in the difference spectra. All of these amino acids are estimated to be within 17 A of the paramagnetic hapten. By using difference spectra involving low fractional occupancy of the combining site with the spin-label hapten, it is established that one threonine and one valine are very close to the paramagnetic hapten.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTMagnetic resonance of a monoclonal anti-spin-label antibodyJacob Anglister, Tom Frey, and Harden M. McConnellCite this: Biochemistry 1984, 23, 6, 1138–1142Publication Date (Print):March 13, 1984Publication History Published online1 May 2002Published inissue 13 March 1984https://pubs.acs.org/doi/10.1021/bi00301a016https://doi.org/10.1021/bi00301a016research-articleACS PublicationsRequest reuse permissionsArticle Views73Altmetric-Citations48LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts

The nuclear magnetic resonance spectra of an Fab fragment of a monoclonal antibody specifically directed against a nitroxide spin-label hapten have been recorded at different concentrations of the hapten. The hybridoma producing this antibody was grown on deuterated phenylalanine, tryptophan, and 3,5-dideuteriotyrosine or 2,6-dideuteriotyrosine. Difference spectra--without hapten minus with hapten--were calculated for each concentration of hapten. The difference spectra reveal five well-resolved singlet proton resonance signals from tyrosine deuterated in the 3,5-positions (H 2,6 Tyr) and nine from tyrosine deuterated in the 2,6-positions (H 3,5 Tyr). The measured intensities of these signals as a function of combining site occupation have been interpreted in terms of a theory involving intrinsic line widths (T2), the hapten off-rate (k), and distances to the paramagnetic center. Good agreement with theory is found for all of the isolated proton signals. The best estimate of k is 350 s-1; distances in the range 13 to less than 9 A are calculated. Extension of this analysis to other amino acids is discussed.

Natural killer (NK) cells have been obtained from mouse spleens and grown in vitro. These cells: retained cytotoxicity against YAC targets; and were homogeneous as judged by morphology and surface markers. The alpha-naphthol acetate esterases (ANAE) and diisopropyl-fluorophosphate (DFP)-binding proteins of NK cells, YAC cells and NK-YAC conjugates have been examined. Ultrastructural cytochemistry indicates that NK cells have two types of ANAE: an enzyme primarily associated with the granule externum and an activatable ANAE associated with the granule internum. Both of these activities are inhibited by DFP. The activatable ANAE appears during incubation of NK cells with YAC cells. DFP-binding proteins were examined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis and autofluorography. NK cells and YAC cells have major DFP-binding proteins of 35 X 10(3) and 20 X 10(3) Mr, respectively. NK-YAC conjugates had a new band at 55 X 10(3) Mr. This new protein may be identical to the activatable ANAE described above. Our studies provide evidence for an NK cell esterase that becomes activated when incubated in the presence of tumour cells. This esterase may be related to the cytolytic mechanism.

Natural killer (NK) cells were obtained from C3H mouse spleens according to a modified version of the method of Kuribayashi et al. [Kuribayashi, K., Gillis, S., Dern, D. E. & Henney, C. S. (1981) J. Immunol . 126, 2321-2327]. These cells retain in vitro cytotoxicity against certain model tumor cell targets and appear homogeneous by morphological criteria. NK cells, YAC (tumor) cells, and NK cell-YAC cell conjugates have been examined with scanning (SEM) and transmission (TEM) electron microscopy. SEM experiments have shown that: ( i ) NK cells are large and possess various shapes in contrast to the YAC target cells which are smaller and round, ( ii ) YAC cells have uniformly distributed microvilli whereas the NK cell microvilli are most prominent in the area of effector-to-target contact, and ( iii ) in the absence of target cells, NK cell microvilli are found in a small number (usually 1-3) of cell surface locations. The region of NK cell-tumor cell contact has also been examined with TEM. The cells were stained with ruthenium red/OsO 4 . The electron-dense ruthenium red/OsO 4 reaction product was consistently found in regions of close cell-cell contact, suggesting that carbohydrates were not completely cleared from areas of contact and that target and effector membranes do not fuse extensively. TEM observations indicate that NK cells have structurally unique granules. The granules are composed of at least two distinct compartments. The outer compartment contains the lysosome-associated enzymes acid phosphatase and inorganic trimetaphosphatase. No enzymatic activities have been found associated with the inner compartment. NK cells appear to degranulate when incubated with YAC cells. Under those circumstances, limited areas of the NK cytoplasm contain vacuole-like areas possessing granules and apparent granular debris. Degranulation appears to be involved in the cytotoxic function of NK cells.

32P labeling of microtubular protein by endogenous protein kinase activity is shown to result from a net increase in protein-bound phosphate and is not the result of a phosphate exchange reaction between ATP and phosphoprotein. Protein phosphorylation is maximal in the presence of 0.5 mM Mg2+ and 0.25 mM ATP, resulting in approximately 2.8 nmol of phosphate/mg of protein. However, phosphorylation can be increased two-to threefold by cAMP. The protein substrates for phosphorylation either the absence or presence of cAMP are the microtubule-associated proteins which copurify with tubulin and promote microtubule assembly. Phosphorylation of microtubule-associated proteins inhibits both the rate and extent of microtubule assembly when the protein is exposed to conditions which result in dissociation of rings. These results are taken to indicate that phosphorylation modifies MAPs so that they have a reduced ability to form an assembly-competent complex with tubulin.