Flow cytometric analysis of the effect of activated macrophages on alveolar cell carcinoma of lung in vitro

Evidences have shown that activated macrophages were cytotoxic to tumor cells but the mechanism of this killing effect is not well understood. Flow cytometry was used to study the effect of activated macrophages on cell proliferation cycle of alveolar cell carcinoma of lung (A 549). Among 74 C57BL/6J mice, 30 as experimental group were given corynebacterium parvum 0.5 ml (1 mg)/mouse intraperitoneally, and 44 as control group were given normal saline 0.5 ml/mouse instead. Peritoneal macrophages of both groups were co-cultured with A 549 in the ratio of 10:1 and 20:1 for 24 and 48 hrs respectively. Specimens were assayed with LXJ-Laser flow cytometer and relative % of tumor cells in G1/O, S and G2 + M phased were calculated by morphometry using Baisech's mathematical model and Zeiss OPTON Video plan. Results indicated that in the experimental group with E/T 20:1, co-cultured for 24 hrs., the % of tumor cells in S phase was markedly decreased (28.44%) and those in G1/0 phase was increased (60.18%) (P less than 0.01); while those of the control group were 40.41% and 49.99% respectively. Changes present in S and G1/0 phase tumor cells of the experimental group indicate that activated macrophages may block tumor cells from the G1/0 to S phase and thus affect their proliferation.     

Pulmonary interstitial macrophages: isolation and flow cytometric comparisons with alveolar macrophages and blood monocytes

Pulmonary interstitial macrophages (IM) were isolated from rat lungs by an Fc gamma receptor-based affinity technique coupled with multiparameter flow cytometry. Single cell suspensions obtained by collagenase digestion of extensively perfused and lavaged lungs were applied to carpets of opsonized sheep red blood cells (SRBC-IgG) bound to plastic tissue culture flasks. At 0-4 degrees C, optimal binding of lung cells occurred within 60 min at plating densities of 1-2 X 10(6) lung cells/cm2 when the SRBC substrate was opsonized with 10 micrograms/ml anti-SRBC IgG. Nonadherent cells were removed by gently rinsing the plates and adherent cells were recovered by lysing the SRBC-IgG substrata. By light microscopy, the mixture of adherent cells was comprised of mononuclear cells (approximately 54%), many of which appeared to be macrophages, lymphocytes (approximately 20%), polymorphonuclear leukocytes (approximately 15%), plasma cells (approximately 8%), eosinophils (approximately 2%), and mast cells (approximately 0.5%). The cells which adhered to the SRBC-IgG monolayers were further resolved into subpopulations by multiparameter flow cytometry and sorted according to their electro-optical characteristics. One subpopulation appeared morphologically to be macrophages, and greater than 90% of these cells readily phagocytized SRBC-IgG in vitro. Peroxidase staining of this population was minimal, indicating that these cells were not blood monocytes (BM). Using a method by which alveolar macrophages (AM) were prelabeled with SRBC-IgG in situ, we demonstrated that alveolar macrophages constituted only approximately 5% of the total adherent cell population. We concluded from these observations that the macrophage population harvested in this manner were neither BM nor AM, but, rather, were harvested from the lung's interstitial compartment. Flow cytometric analyses indicated that the IM exhibited electro-optical characteristics intermediate between those of BM and AM, which is consistent with the concept of the lung's interstitium as a maturation compartment for the BM prior to migration into the alveolar compartment. However, the IM more closely resembled the BM than the AM, indicating that if the IM is in fact a precursor to the AM population, substantial maturation or differentiation must occur subsequent to its migration into the alveolar compartment. This isolation technique will be useful for harvesting highly purified IM for in vitro investigations.     

Flow cytometric analysis of PKH26-labeled goldfish kidney-derived macrophages

We recently demonstrated that a goldfish macrophage cell line (GMCL) and primary in vitro-derived kidney macrophage (IVDKM) cultures contain three distinct macrophage subpopulations. Morphological, cytochemical, functional, and flow cytometric characterization of these sub-populations suggested that they may represent cells of the macrophage lineage temporally arrested at distinct differentiation junctures of fish macrophage development (putative early progenitors, monocytes, and macrophages). In this study, we examined the proliferation and differentiation events leading to the generation of mature macrophage-like cells from goldfish kidney hematopoietic tissues. The flow cytometric studies were done after labeling macrophages with PKH26 fluorescent dye and analysis of the data using the MODFIT software. Our results showed that IVDKM cultures proliferated non-synchronously, suggesting the presence of a temporal control mechanism regulating the number of cells entering the paths towards maturation. Such control is most evident during early progenitor proliferation and differentiation events. Our results showed that proliferation may not be a requirement for differentiation of early progenitors to putative monocyte and macrophage subsets. Detailed observation of the mature macrophage-like subpopulation indicated that: 1) they appear to develop from both, the differentiation of monocyte-like cells, and direct differentiation of early progenitors in the absence of a monocyte-like stage; and (2) mature macrophage-like cells appeared to be capable of self-proliferation. Our results suggest the presence of alternate pathways of fish macrophage development other than the classical hematopoietic pathway.     

Flow cytometric analysis of I-J expression on murine bone marrow-derived macrophages

Attempts to analyze bone marrow-derived macrophages (BMDM) by flow cytometry have been prohibited because of their high autofluorescence. Using an autofluorescence reduction method of Steinkamp and Stewart to reduce the autofluorescence of BMDM, we were able to examine several macrophage populations for their expression of I-A, I-J, and Mac-1 cell surface determinants. Bone marrow cells examined immediately after removal from the femur contain 50-60% Mac 1-positive cells (mainly granulocytes). During the next few days granulocytes and nonmacrophage precursor cells die, and the number of Mac 1-positive cells decrease. Once the bone marrow cells have been maintained in L cell conditioned medium (LCM) for 2 to 3 days, the number of cells expressing Mac 1 increases rapidly from 20% to 98% during the next 3 to 4 days. Bone marrow cells grown in LCM do not express I-J until these cells have been in culture 3 to 4 days, and the number of cells expressing I-J (up to 90% positive) parallels the increase in macrophages. Bone marrow cells maintained in LCM did not express detectable I-A during the 14 days these cells were examined. Two other macrophage populations often used in a variety of immunological studies were analyzed by flow cytometry. We found that the majority (up to 80%) of peritoneal cells expressed I-A, and only 20% of peritoneal cells had I-J cell surface determinants. On the other hand, peritoneal exudate cells collected 4 days after thioglycolate medium treatment were predominantly I-J positive (up to 70%), and only about 30% of these cells expressed I-A cell surface antigens. The binding of anti-I-J IgM antibody to BMDM was not to Fc receptors because pretreating these cells with up to 25 micrograms of an IgG2a myeloma protein did not block anti-I-J antibody binding. The addition of 25-200 micrograms of monoclonal anti-Fc receptor antibody was also ineffective in blocking the binding of a monoclonal anti-I-Jk antibody to BMDM. Pretreatment of BMDM with the IgM fraction of several control IgM antibody preparations did not block the specific binding of fluoresceinated anti-I-J IgM antibody. BMDM provide a pure population of macrophages that express a significant level of cell surface I-J antigens. Bone marrow cells grown in LCM are essentially devoid of other contaminating cells, and the increase in the number of I-J-positive cells parallels the increase in macrophages in these cultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Flow cytometric characterisation of cell populations in human pregnancy decidua and isolation of decidual macrophages

Methods have been developed for isolating human tissue macrophages from first trimester or term pregnancy decidua. After a two stage enzymic digestion, viable cells were separated from cellular debris by velocity sedimentation at unit gravity or by Percoll centrifugation. Cell populations were analysed by flow cytometry after labelling with monoclonal antibodies. In term decidua, 47% of the cells were of bone marrow origin, comprising 18% macrophages, 3% large granular lymphocytes and 8% T cells. The remaining cells, the proportion of which varied between individuals, were CD16-positive granulocytes. Macrophages were isolated flow cytometrically from both first trimester and term decidual cell dispersions after labelling with an antibody to MHC class II. Yields of up to 4 X 10(6) macrophages, greater than 95% pure, were routinely obtained.     

Flow cytometric leucocyte counts

A flow cytometric method was evaluated for performing total leucocyte counts on bovine blood. Fifty blood samples from 19 healthy Holstein cows were analysed on a flow cytometer. The method involved diluting blood with either hypotonic or isotonic saline solution, lysing the red blood cells, and performing a 2-parameter analysis on the basis of cell size and cellular granularity. Leucocyte numbers were determined by creating a window on dot plots of cell size (determined by forward light scatter) vs cellular granularity (determined by the logarithm of side light scatter). Total leucocyte counts determined by flow, cytometer with either diluent were lower (p<0.01) than counts determined by use of an electronic particle counter. The correlations between electronic particle counts and flow cytometric counts using isotonic or hypotonic diluents were 0.857 and 0.458, respectively. The determination of total leucocyte counts using flow cytometry of a blood sample diluted in isotonic saline and treated to lyse red blood cells shows potential as a way for counting leucocytes.     

Flow cytometric analysis of lymphomas

Flow cytometry is rapidly developing as an important tool in the characterization of lymphomas. Analysis of cell surface antigens and DNA content in these tumors provides useful biologic information that can be applied advantageously to their diagnosis and classification. In this article we examine practical aspects of handling, preparation, and staining of lymphoid samples for flow cytometry and delineate approaches to data analysis and interpretation. We discuss specific applications, advantages, and limitations of the technique in the evaluation of neoplastic lymphoid diseases.     

Flow cytometric detection of viruses

Representatives from several different virus families (Baculoviridae, Herpesviridae, Myoviridae, Phycodnaviridae, Picornaviridae, Podoviridae, Retroviridae, and Siphoviridae) were stained using a variety of highly fluorescent nucleic acid specific dyes (SYBR Green I, SYBR Green II, OliGreen, PicoGreen) and examined using a standard flow cytometer equipped with a standard 15 mW argon-ion laser. The highest green fluorescence intensities were obtained using SYBR Green I. DNA viruses with genome sizes between 48.5 and 300 kb could easily be detected. The fluorescence signals of the small genome-sized RNA viruses (7.4-14.5 kb) were found at the limit of detection. No significant linear relationship could be found between genome size and the green fluorescence intensity of the SYBR Green I stained virus preparations. To our knowledge, this is the first report of detecting and discriminating between a wide range of different viruses directly using flow cytometry. This rapid and precise assay represents a new and promising tool in the field of virology.     

Flow cytometric studies of anti-granulocyte antibodies

An indirect immunofluorescence test using anti-granulocyte antibodies was developed to examine many sera samples in a one-step procedure by flow cytometry. Granulocytes obtained from several random donors were fixed with 0.5% paraformaldehyde solution on the first day, and the fluorescence intensity of granulocytes that had reacted with sera was measured on the following day. Non-specific reactions with isoantibodies, i.e., anti-A, anti-B, or serum IgG had no influence on the fluorescence intensity. Therefore, anti-granulocyte antibodies from a patient with immune neutropenia and patients with febrile transfusion reactions can be studied using this method. The results suggest that the IgG-antibody from a patient with immune neutropenia is granulocyte-specific and its subclass is IgG2.     

Flow cytometric measurement of intracellular cytokines

The identification of distinct T helper lymphocyte subsets (Th1/2) with polarised cytokine production has opened up new fields in immunobiology. Of the several alternative methods of monitoring cytokine production, flow cytometric analysis of intracellular staining has distinct advantages and pitfalls. It allows high throughput of samples and multiparameter characterisation of cytokine production on a single cell basis without the need for prolonged in vitro culture and cloning. However, these methods may cause important changes in cell surface phenotype which can make interpretation difficult.     

Defective chemotaxis of human alveolar macrophages

Human pulmonary alveolar macrophages (PAM) from normal subjects, unlike peripheral blood monocytes (PBM), are unable to migrate in response to various chemoattractants, such as C5a,f-Met-Leu-Phe (fMLP) and phorbol myristate acetate (PMA). Inflammatory PAM obtained from sarcoid patients also failed to exhibit a chemotactic response. Binding studies using [3H]PDBU demonstrate high affinity receptors for phorbol esters on PAM surface, in a comparable amount (1.5-2.4 X 10(6) receptors/cell) to PBM (8-15 X 10(5) receptors/cell). Moreover, PAM were comparable to PBM in terms of superoxide anion (O2-) release in response to PMA. Therefore, the defective locomotory response of PAM cannot be accounted for by lack of chemoattractant receptors, at least for phorbol esters. Worthy of note, PMA receptors on PAM are able to transduce activating signals for O2- generation. These findings show that competence for chemotaxis is heterogeneously distributed among mononuclear phagocytes.     

The prolonged life-span of alveolar macrophages

To further examine the half-life of alveolar macrophages, chimeric CD 45.2 mice were generated through bone marrow transplantation of donor CD 45.1 cells. Before administration of donor cells, recipient mice were divided into two cohorts: the first cohort received total body irradiation; the second cohort also received irradiation-however, the thorax, head, and upper extremities were shielded with lead. Flow cytometric analysis was then performed on blood, peritoneal, and bronchoalveolar lavage cells over time to quantify engraftment. The data generated for the unshielded cohort of mice revealed a macrophage half-life of 30 days. In the shielded cohort, however, we found that by 8 months there was negligible replacement of recipient alveolar macrophages by donor cells, despite reconstitution of the blood and peritoneum by donor bone marrow. Consistent with these findings, the mean fluorescent intensity of alveolar macrophages remained stable over a 4-week period after in vivo PKH26 dye loading. Together, these data show that previous alveolar macrophage half-life studies were confounded by the fact that they did not account for the toxic effects of irradiation conditioning regimens, and demonstrate that the bone marrow does not significantly contribute to the alveolar macrophage compartment during steady-state conditions.     

Physiologic regeneration of alveolar macrophages]

Alveolar macrophages (AM) play one of the leading roles in the modification of structure, function and behaviour of the respiratory organs cells. The size of AM population may determine the expression of the inflammatory and regenerative processes in the lung. Restoration of AM population may be brought about by two routes from monocytes coming to the alveoli from the circulation with subsequent differentiation into AM and as a result of local monocytes and macrophages proliferation. AM is a long-living cell population capable for limited self-maintenance and renewal.     

Flow cytometric analysis of bromodeoxyuridine-induced micronuclei

The effects of DNA substitution by the thymidine analogue 5-bromodeoxyuridine(BrdU) on cell cycle progression and micronucleus inductionwere studied in different mammalian cell cultures. Simultaneousflow cytometric measurements of DNA content and side scatterof nuclei in Chinese hamster embryo (CHE) cells revealed a concentration-dependenttemporary block in the G₂/M phase of the first cell cycle. NTH3T3 cells and human amniotic fluid fibroblast-like cells, onthe contrary, did not show any cell cycle disturbances in thepresence of BrdU. Micronucleus frequency increased as soon asCHE cells started to divide and reached a plateau when all cellshave divided. The height of this plateau was almost equal for60 and 100 µM BrdU. This saturation of micronucleus inductionwas due to a saturation of BrdU incorporation into DNA alreadyat a dosis of 60 µM as shown by the BrdU/Hoechst quenchingtechnique. Indirect immunofluorescent staining of kinetochoreswith CREST antibodies revealed that nearly all BrdU-inducedmicronuclei were kinetochore-negative suggesting the presenceof acentric chromosome fragments in these micronuclei. DNA distributionsof micronuclei measured by flow cytometry showed several peaksrepresenting micronuclei which contain DNA fragments of definedsizes induced by non-random breakage of chromosomes 1 and Xas verified by flow karyotyping and C-banding.     

Flow cytometric measurement of antiplatelet antibodies

A flow cytometric technic was developed to detect platelet surface-bound immunoglobulin in patients with thrombocytopenia. Elevated platelet surface IgG and/or IgM was detected in 90.9% of patients with immune thrombocytopenia purpura (ITP). False positive results occurred in 9.3% of patients with nonimmune thrombocytopenia usually associated with sepsis. False negatives occurred most frequently in adults with chronic ITP. Measurement of platelet surface immunoglobulin with this flow cytometric technic helps differentiate immune from nonimmune thrombocytopenia.     

Flow cytometric analysis of stemline heterogeneity

The stemline heterogeneity of malignant tumors is closely connected with tumor aneuploidy. Both features can be characterized by either chromosome analysis or DNA cytophotometry. DNA analysis may be performed either by single cell photometry or by flow cytometry, of which the respective advantages and drawbacks are presented. Preliminary results of flow cytometric DNA analysis in malignant neoplasms are discussed; the possibilities of multiparametric measurement for quantitative analysis of various biochemical and antigenic properties in DNA stemlines are considered.