Simultaneous analysis of cell surface antigens, bromodeoxyuridine incorporation and DNA content

A method has been developed for correlating the presence of cell surface markers with bromodeoxyuridine (BrdUrd) uptake. We have found that paraformaldehyde fixation will maintain immunofluorescent cell surface staining through acid denaturation that is necessary for anti-BrdUrd reactivity to single-stranded DNA. In addition, the forward angle light scattering was maintained, even though the cells had been exposed to 2N HCl and detergent. The protocol was tested on three model systems: mouse thymus, a human cell line (CCRF-SB), and peripheral blood leukocytes. It was found that there was no specific loss of lymphocyte subsets.     

Cell surface characteristics and DNA content of macrophages in murine bone marrow cultures

Summary An instrument combining scanning electron microscopy (SEM) and light microscopy (LM) was used to study the cell surface characteristics and DNA content of macrophages in murine bone marrow cultures. After a quantitative Feulgen DNA staining, the DNA content of the individual macrophages was measured and their cell surface morphology was studied immediately thereafter with the SEM part of the instrument. The cells were divided into six groups according to the number of microvilli and/or microridges present on their surface. A proportion of macrophages showed a DNA content more than occurs in diploid cells, which could indicate a future division. No special surface morphology could be detected in this cell type.     

Simultaneous cytofluorometric analysis for the expression of cytoplasmic antigens and DNA content in CD3- human thymocytes

We describe a method of two-color immunofluorescence staining which allows the simultaneous analysis of both cytoplasmic antigens and cell entry into the S/G2/M cell cycle phases. This analysis was performed on CD3(-)-activated thymocytes obtained from either highly purified CD1-CD3-CD4-CD8- cells or fresh thymus cell suspensions, stimulated with low doses of phorbol-12 myristate-13 acetate (0.5 ng/ml) and interleukin-2. On the 14th day under these culture conditions about 90% of thymocytes did not express CD3 antigen on the cell surface. CD3- cells were further purified by cell sorting, fixed in paraformaldehyde, and permeabilized with Nonidet-P40. Then these thymocytes were stained by indirect immunofluorescence with monoclonal antibodies identifying T cell-specific molecules (CD3, CD2, CD28, TCR alpha/beta, and TCR gamma/delta) and analyzed for DNA content. Interestingly, both CD3 and CD28 antigens were detectable in the cytoplasm of most cells (greater than 80%). Further, the majority of the thymocytes which had entered the S/G2/M phases of the cell cycle (20%) expressed intracellular CD3 and CD28 molecules and reacted with the anti-beta framework beta F1 monoclonal antibody. The relationship between the appearance of CD3 and other T cell markers in the cytoplasm, the cell cycle entry, and the thymocyte development is discussed.     

Simultaneous measurement of DNA content and detection of surface antigens of cervicovaginal cells by flow cytometry

A method is described by which the measurement of the DNA content and the light scatter and the detection of a cervical carcinoma-associated antigen (CCA) of squamous epithelial cells can be simultaneously accomplished by flow cytometry (FCM). Cervicovaginal cellular samples obtained from 30 patients were analyzed by this method. Cell populations with an abnormal DNA content or with the presence of CCA were detected in 20 samples, 18 of which contained dysplastic cells as detected by routine cytologic screening. The remaining ten cases, which were interpreted as cytologically normal by routine screening, were also interpreted as normal by FCM analysis.     

Simultaneous measurement of DNA content and cell-surface immunofluorescence of human bone marrow cells using a single laser flow cytometer.

This paper describes the measurement of S phase DNA content in human bone marrow subpopulations using a single laser method for bivariate analysis of DNA content and cell-surface immunofluorescence (s-IF). Low density (less than 1.077 g/ml) bone marrow cells were labeled with a panel of unconjugated monoclonal antibodies (MoAb) for the lymphoid (CD2 + CD19), T-lymphoid (CD2), B-lymphoid (CD19), erythroid (anti-glycophorin-A), myelomonocytic (CD13, CD33; single and as cocktail) and monocytic (CD14) lineages. A fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse label was used as second step. Unfixed, MoAb-labeled cells were incubated for 24 h with a hypotonic propidium iodide solution for DNA staining. Cells were analysed on a single-laser flow cytometer, operating at 488 nm. The effect of the combined staining protocol upon both s-IF and DNA stainability was evaluated. Only a slight decrease (mean: 29.0%) in s-IF intensity was observed after DNA staining. The percentages of immunofluorescent cells in the bone marrow samples of 10 normal individuals before and after DNA staining were essentially unchanged for all the MoAbs used. The DNA histograms of the immunophenotypically defined subpopulations were of excellent quality with a mean coefficient or variation of 1.8%. This procedure allows the assessment of very low levels of S-phase DNA content, as measured in normal low density blood cells of 8 healthy volunteers (mean 0.07%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Simultaneous paired analysis by flow cytometry of surface markers, cytoplasmic antigens, or oncogene expression with DNA content

Flow cytometry is a useful tool for measuring DNA content and differentiation as expressed by cell surface markers. We have extended this technology to measure simultaneously either surface, cytoplasmic, or nuclear antigens (particularly oncoproteins) with DNA content. Mononuclear blood cells isolated from normal subjects and HL60 leukemic cells were permeabilized and fixed in suspension utilizing 40 micrograms/ml lysolecithin and 1% paraformaldehyde. A range of lysolecithin concentrations in 1% paraformaldehyde was studied to optimize permeabilization of the antibodies to the cell interior without destroying cell integrity. The optimal concentration (40 micrograms lysolecithin/ml) resulted in good cell recovery with a high percentage of cells positive for surface and intracellular antigens. Cells are first stained with fluorescein isothiocyanate conjugated (FITC) antimyeloperoxidase (an azurophil granule enzyme), or with an anti-c-myc antibody and FITC goat anti-mouse IgG F(ab')2. Cells are then incubated with RNase and stained for DNA content with propidium iodide. Alternatively, cells were stained for the cell surface markers Leu M3, OKM1, or the transferrin receptor and were then fixed and permeabilized and stained with propidium iodide. Using this method, we correlated cytoplasmic, nuclear, or cell surface antigens with cell cycle kinetics. This technique should be useful for studies of cellular differentiation and proliferation.     

Immunodiffusion analysis of water-soluble rat bone marrow antigens

Antisera were obtained against six electrophoretic fractions of the rat bone marrow extract. With their help, 18 tissular antigens and 11 antigens immunologically similar to the blood serum proteins were revealed in the rat bone marrow. All tissular antigens are divided in five groups by the degree of organ specificity: 1) bone marrow organospecific antigens (4 antigens), 2) antigens present in the bone marrow, spleen and lung extracts (2), 3) "granulocytic" antigens (4), 4) antigens common for many rat organs, but not found in the extracts of blood formed elements, skeletal muscle, heart, brain, eyes (3), 5) antigens present in all the organs studied (5). The bone marrow organospecific antigens may be specific antigens of hemopoietic cell precursors. The possibility of utilization of antisera against the bone marrow water soluble proteins for labelling hemopoietic cells of different lines of differentiation is discussed.     

Immunoperoxidase detection of myeloid antigens in glycolmethacrylate-embedded human bone marrow

Different methods for fixation and exposure of antigenic determinants were tested for detection of a granulocytic differentiation antigen by the monoclonal antibody L12-2, using an indirect immunoperoxidase method on semi-thin sections of undecalcified, glycolmethacrylate-embedded human bone marrow biopsies. Fixation in Bouin's solution for 3 hr gave a more intense and more homogeneous immunological staining than fixation in absolute methanol, 4% formalin, B5, or Michel's medium, and the morphological detail was excellent. Digestion by pronase or trypsin was required. Coating the glass slides with Alcian blue prevented loss of sections from the slides during the staining procedure. Bouin fixation also made possible detection of two other differentiation antigens expressed in the granulocytic series, using the monoclonal antibodies 1G10 and R1B19. Furthermore, the same technique also permitted detection of factor VIII-RAg in the megakaryocytes, as well as recognition of cells of the erythroid series by use of polyclonal rabbit antisera.     

Simultaneous flow cytometric analysis of two cell surface markers,telomere length,and DNA content

BACKGROUND: Various protocols for estimation of telomere length in individual cells by flow cytometry using fluorescence in situ hybridization of fluorescently labeled peptide nucleic acid (PNA) probes (Flow-FISH) have been described. Combined analysis of telomere length and cell phenotype, however, remains difficult because few fluorochromes with suitable emission spectra tolerate the harsh conditions needed for DNA denaturation during hybridization of the telomere-specific PNA probe. We overcame these problems and developed a method for measuring telomere length in cell subsets characterized by the expression of two surface antigens. METHODS: Alexa Fluor 488 and Alexa Fluor 546 were used for cell surface staining. Antigen-antibody complexes were covalently cross-linked onto the cell membrane before Flow-FISH. Cells were hybridized with a PNA probe conjugated to cyanine 5 (Cy5). Hoechst 33342 (HO342) was added for determination of cellular DNA content. For assay standardization, we added an aliquot of a single batch of 1,301 cells to each sample as an internal control before hybridization with the PNA probe. Samples were prepared in duplicate and analyzed on a standard three-laser BD LSR flow cytometer. For assay validation, the same samples were analyzed in parallel to correlate the percentage of telomere length of the sample versus 1,301 control cells to the mean size of terminal restriction fragments (TRFs) of DNA as determined by Southern gel analysis. RESULTS: The method permitted clear identification of lymphocyte subsets in samples hybridized for Flow-FISH, with subset frequencies comparable to those of untreated samples. At a concentration of 10 nM, the Cy5-labeled telomere-specific PNA probe produced a bright fluorescence signal well separated from background. Addition of HO342 in low concentration did not interfere with Cy5 telomere fluorescence, produced adequate DNA histograms, and permitted clear identification of cell phenotype. The probe concentration of 10 nM also proved optimal for inclusion of 1,301 control cells for assay standardization. Telomere length estimations by the current method correlated highly with TRF calculations by Southern gel hybridization (r(2)= 0.9, P = 0.0003). Application of our protocol to the analysis of human CD8CD28 lymphocyte subsets showed that CD8(+bright)CD28(-) lymphocytes generally exhibit shorter telomeres than CD8(+bright)CD28(+) cells. These data concurred with previous results of telomere shortening in CD8(+)CD28(-) T cells that were obtained by using different techniques. CONCLUSIONS: The multiparameter Flow-FISH protocol permitted rapid determination of differences in telomere length in subpopulations characterized by two surface markers without prior cell separation.     

Phenotypic analysis of human bone marrow macrophages

Bone marrow macrophages have been isolated for phenotypic analysis by their ability to bind erythroblasts in erythroid clusters. In situ, when labeled for CD68 antigen, they are seen to form an arborizing network uniformly distributed throughout the hemopoietic marrow. Marrow macrophages isolated within erythroid clusters are acid phosphatase and alpha-naphthol butyrate esterase positive. Immunophenotypically, they are highly reactive for CD4, phagocytic receptors FcRI, II, and III, for HLA-Dr and CD31, as well as for the integrins CD11a, CD11c, and CD18, but negative for CD35 and transferrin receptor epitopes. Comparison of their phenotype with blood monocytes and cultured macrophages reveals significant differences, which indicate that marrow macrophages are specialized, differentiated mononuclear phagocytes that selectively associate with developing erythroblasts.     

Effect of exogenous DNA on the content and biosynthesis of endogenous DNA in rat bone marrow]

The influence of exogenous DNA on the content of endogenous DNA and the rate of biosynthesis was studied in rat bone marrow. After the injection of highly-polymerized homologous DNA to intact rats the content of rat DNA per 1 gm of the bone marrow decreased within the first 3 days (the most marked fall occurred in 3 days--by 36%), and returned to the normal by the 6th day. The rate of DNA biosynthesis in rat bone marrow increased in 18 hours (doublled in comparison with control), remained elevated within 6 days (by 58%) and approached the normal level from 1 to 3 days after the DNA injection.     

Carbohydrate and peptide antigens in macrophage populations derived from human bone marrow and milk: an immunomorphological and immunochemical analysis

Summary An immunomorphological and immunochemical study was performed to elucidate the pattern of carbohydrate antigens and their relationships to the cluster differentiation (CD) 68 epitopes on macrophages derived from human bone marrow and milk. Core and backbone antigens recognized by lectins fromBauhinia purpurea (BPA),Helix pomatia (HPA),Arachis hypogaea (PNA),Glycine max. (SBA),Griffonia simplicifolia (GSA-I-B₄),Lycopersicon esculentum (LEA) andErythrina cristagalli (ECA) were expressed by both macrophage populations. Additionally, they exhibited various peripheral type 1 and type 2 carbohydrate antigens. In bone marrow trephine biopsies, the number of macrophages stained by the CD68-specific monoclonal antibody PG-M1 exceeded significantly (range 30–40%) the subpopulation expressing SBA, GSA-I-B₄ and ECA binding sites as well as the Lewis^a antigen. This result is very interesting since, fromin vitro studies, GSA-I-B₄ and SBA are known to react especially with activated macrophages. Western blotting experiments on milk macrophage lysates revealed that ECA, GSA-I-B₄, BPA, PNA and MAA visualize a 110 kDa band isographic with the CD68 antigen detected by PG-M1, KP1 and Ki-M1P monoclonal antibodies. These antibodies recognize peptide epitopes as shown by enzyme-linked immunosorbent assays after biochemical modification of milk macrophage lysates. This result is in keeping with the assumption that the CD68 antigen consists of a highly glycosylated mucin-type glycoprotein comprising various differentiation-dependent epitopes.     

Simultaneous cytometric analysis for the expression of cytoplasmic and surface antigens in activated T cells

A method of two-colour immunofluorescence staining has been developed to allow the simultaneous analysis of both surface and cytoplasmic antigens. This involves the use of direct fluorochrome antibody conjugates for cell-surface antigen staining, followed by cell permeabilization and the staining of cytoplasmic antigens with biotinylated antibodies and streptavidin-fluorochrome conjugates. Fluorochrome-antibody conjugates bound to cell-surface epitopes were found not to be affected by the subsequent permeabilisation and cytoplasmic staining. This method was used to examine the surface phenotype of T cells expressing a cytoplasmic antigen, STA. STA is a unique determinant detected in activated human T cells by the monoclonal antibody K-1-21, which also recognizes a cross-reactive conformation-dependent epitope on human free kappa light chains. Cytometric analysis showed that STA is found in both Leu 2a+ cytotoxic/suppressor T cells and Leu 3a+ helper/inducer T cells but is not induced in the Leu 15+ population which contains suppressor T cells. STA was also shown to be an activation antigen in murine T cells.     

Blood volume and haemoglobin oxygen content changes in human bone marrow during orthostatic stress

The interest in, and the need for effective measures to be used in screening, diagnosis, and the follow-up of skeletal pathologies is growing markedly. This paper proposes a completely new and non-invasive technique allowing the study of the human tibia bone marrow (BM) haemodynamics with a time resolution of 1 s. The technique, based on near infrared spectroscopy, is sensitive enough to allow the detection of BM blood volume and/or oxygen saturation changes during orthostatic variations imposed by a tilt bed. An increase in the slope of the bed of 15 degrees is sufficient to detect this phenomenon. The ability to study the possible presence of a neural control of BM haemodynamics is also discussed. No other existing technique currently allows one to obtain the proposed results and this approach might open up a new field of study related to human BM physiology.     

Insights into the expression of ABH and Lewis antigens through human bone marrow transplantation.

Twelve information bone marrow transplants, with at least one difference in ABO and/or Lewis types between donor and recipient, were retrospectively studied. ABH and Lewis antigens were determined in plasma, erythrocytes, and lymphocytes. Donor lymphocytes acquired the ABH and Lewis antigens from the recipient's plasma in the same way that donor erythrocytes acquired the Lewis antigens from it. Lymphocytotoxicity detected type 1 ABH and Lewis antigens only, providing evidence for the existence of combined ABH and Lewis antigens on lymphocytes. This was in contrast with the ABH antigens on type 2 chains of red cells, which are devoid of Lewis specificities. The differences in genetic control, probable chemical structure, and cellular origin of these two types of ABH antigens are presented in a theoretical model that accounts for most of the known data.