The Hoechst low-fluorescent profile of the side population: clonogenicity versus dye retention.
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Animals↗
Biomedical subjects
Pedro Marin
Publications and source records attributed to Pedro Marin.
Flow cytometry of the Side Population: tips & tricks.
BACKGROUND: The Side Population (SP) has become an important hallmark for the definition of the stem cell compartment, especially in the detection of these cells and in their physical isolation by fluorescence-activated cell sorting (FACS). SP cells are CD34neg and were discovered using ultraviolet excitation based on the efflux of Hoechst 33342 (Ho342). Although the method works as originally described, we believe that this method is difficult for most investigators. First, because the ability to discriminate SP cells is based on the differential retention of Ho342 during a functional assay; second, because of the difficulties in setting the right experimental and acquisition conditions; and third, because the analysis of the acquired data requires an extensive expertise on flow cytometry to accurately detect the SP events. METHODS: First of all and mainly for the SP application, the laser beam paths were exhaustively checked to ensure the lowest coefficients of variation. Blood suspensions were prepared by erythrocyte lysis with ammonium chloride and hematopoietic cells were labeled with Ho342. RESULTS: The Ho342 concentration and the staining procedure are critical for the optimal resolution of the SP cells. Although UV laser alignment is very important to resolve the dim tail that outlines the SP, the problem with Ho342 excitation is not the Hoechst Blue emission, but rather the Hoechst Red's (because of the weak emission). CONCLUSIONS: Each laboratory must establish its own expected ranges based on its instrument and results may vary slightly due to instrument differences such as the narrowness of the band pass filters, laser power, laser emission wavelength, nozzle type, differential of pressure, light collection system (cuvette versus jet-in-air) and beam shaping optics.
Animals↗
A rare fraction of human hematopoietic stem cells with large telomeres.
The lack of specific markers for stem cells makes the physical identification of this compartment difficult. Hematopoietic stem cells differ in their repopulating and self-renewal potential. Our study shows that multiple classes of human hematopoietic CD34+ greatly differ in telomere length. Flow-cytometry-based fluorescent in situ hybridization and confocal microscopy of CD34+ cells has revealed remarkable telomere length heterogeneity, with a hybridization pattern consistent with different classes of human hematopoietic progenitor cells. These results also point to the existence of a significant clonal heterogeneity among primitive hematopoietic cells and provide the first evidence of a rare fraction of CD34+ cells with large telomeres in humans.
Antigens, CD34↗
Flow cytometry-based approach to ABCG2 function suggests that the transporter differentially handles the influx and efflux of drugs.
BACKGROUND: To better characterize the function of the ABCG2 transporter in vitro, we generated three cell lines (MXRA, MXRG, and MXRT) stably expressing ABCG2 after transfection of wild-type ABCG2 and two mutants (R482G and R482T), respectively. METHODS: ABCG2 expression and function were analyzed by flow cytometry using monoclonal antibodies, a variety of fluorescent substrates, and a series of potential inhibitors of the transporter. RESULTS: ABCG2 expression was detected in all cell lines. The cell lines effluxed mitoxantrone (MXR), but only the mutants effluxed rhodamine 123 (Rho123), SYTO13, doxorubicin, and daunorubicin. After incubation with MXR, intracellular accumulations were 9- and 22-fold higher in MXRA than in MXRT and MXRG cells, respectively, suggesting that ABCG2 also modulates the influx rate of the drug. Flow cytometry kinetic studies of MXR efflux showed that MXRG cells effluxed 50% of the drug at a faster rate than MXRA and MXRT cells (t50: 15.3 min vs. 27.8 and 44.5 min, respectively). MXRG cells also extruded Rho123 and SYTO13 at a faster rate than MXRT cells. ABCG2-mediated transport was inhibited by fumitremorgin C, cyclosporine A, and PSC-833, but not by verapamil or probenecid. MXRG cells displayed the highest level of resistance to MXR, doxorubicin, and daunorubicin in the cytotoxicity assays. CONCLUSIONS: Glycine mutations at position 482 have a significant impact on ABCG2 function by modifying its substrate specificity and its influx/efflux rates. This study also demonstrates that flow cytometry constitutes a powerful tool for the kinetic analysis of ABC transporters.
ATP Binding Cassette Transporter, Subfamily G, Mem↗
G-CSF increases the expression of VCAM-1 on stromal cells promoting the adhesion of CD34+ hematopoietic cells: studies under flow conditions.
OBJECTIVE AND METHODS: The knowledge of the mechanisms underlying the adhesive processes that lead to homing and/or mobilization of hematopoietic progenitor cells, and the influence of blood rheology, is still limited. We analyzed the impact of flow conditions on the adhesion of CD34+ peripheral blood progenitor cell (PBPC) to the adhesive proteins fibronectin, laminin, and collagen, and to stromal cells. RESULTS: Under static conditions, all the adhesive substrata assayed promoted adhesion of CD34+ PBPC, being higher on the stromal cells. Under flow conditions, adhesion of CD34+ PBPC was remarkable on stromal cells while insignificant onto the purified proteins. Exposure of stromal cell monolayers to granulocyte colony-stimulating factor (G-CSF) further enhanced PBPC adhesion. This effect correlated with the activation of p38 MAPK and with an increase in the expression of VCAM-1 on stromal cells exposed to G-CSF. In inhibitory assays, both an antibody to the G-CSFR and a specific inhibitor of the p38 MAPK blocked the effects induced by the cytokine. CONCLUSION: Our results provide direct evidence that in stromal cells G-CSF activates the signaling protein p38 MAPK, inducing expression of the adhesion receptor VCAM-1. This mechanism seems to promote adhesion of CD34+ cells on stromal cells and could play a potential role in homing events.
Antigens, CD34↗
Flow cytometric-based isolation of nucleated erythroid cells during maturation: an approach to cell surface antigen studies.
Nucleated red blood cells (NRBCs) are involved in normal physiologic processes, as well as in several malignancies. They are usually counted manually under the microscope. However, blood sample manipulation may be a source of variability and manual counting is imprecise, time-consuming, and subjective. To improve identification of CD45-negative cells, we used a flow cytometry technique that avoids the addition of lysing reagents and stains viable cell nuclei. We applied this method for counting and isolating NRBC subpopulations in whole blood samples, using DNA/RNA viable staining to discriminate nonnucleated erythroid cells and debris. NRBC counts gave 197.95 cells per mm(3) in mobilized peripheral blood samples (1.00%, n = 20), 3897.59 cells per mm(3) in leukapheresis products (3.08%, n = 20), and 765.21 cells per mm(3) in cord blood samples (6.09%, n = 20). Normal bone marrow counts were 5449.42 cells per mm(3) (11.76%, n = 20). Scatter profiles showed three distinct populations, from early to late-stage erythroblasts, consisting of erythroblasts, orthochromatic erythroblasts, and ejected nuclei, as confirmed by Wright-Giemsa staining. In addition, flow cytometry immunophenotyping showed that glycophorin A was expressed dimly on NRBCs during maturation. These findings point to the feasibility of live NRBCs studies, which offer great potential for a wide range of disciplines.
Antigens, Surface↗
A multicolor, no-lyse no-wash assay for the absolute counting of CD34+ cells by flow cytometry.
BACKGROUND: We previously developed a method for counting CD34(+) cells in unlysed whole blood. This method was applied to normal human bone marrow, peripheral blood after mobilization of progenitor cells, leukapheresis products, and cord blood and was validated with two different lyse-no wash methods. However, the main advantage that we described, erythrocyte discrimination using nucleic acid staining, was also the main restriction because additional markers for the immunologic characterization of CD34(+) cells cannot be included. METHODS: We used SYTO-13 and fluorescein isothiocyanate (FITC)-CD45 staining (FL1) instead of SYTO-13 and phycoerythrin (PE)Cy5-CD45 staining (FL3) to leave the third and fourth fluorescence parameters available for further characterization of CD34(+) cells. The new method was validated by applying it to cord blood samples (n = 20). RESULTS: FITC-CD45 antibody gave a 1.7-fold increase in mean fluorescence intensity over SYTO-13 alone. From absolute counts (CD34(+) cells per microliter), we plotted the differences between the methods against their mean, showing that differences fell into acceptable ranges. CONCLUSIONS: No-lyse procedures may represent an advance for cell immunophenotyping and it could be applied to the measurement of additional markers. Cytometry (Clin. Cytometry) 50:249-253, 2002.
Antigens, CD34↗
Outcome of pregnancy and disease course among women with aplastic anemia treated with immunosuppression.
BACKGROUND: Aplastic anemia may develop during pregnancy and sometimes improves spontaneously after delivery. The effects of pregnancy on aplastic anemia after immunosuppressive treatment and of aplastic anemia on the outcome of pregnancy have not been described. OBJECTIVE: To determine the outcome of pregnancy and the disease course among women with aplastic anemia who received immunosuppressive therapy. DESIGN: Retrospective multicenter study. SETTING: Twelve centers participating in the European Group for Blood and Marrow Transplantation. PATIENTS: 36 women who received immunosuppressive therapy to treat aplastic anemia. MEASUREMENTS: Outcomes of pregnancy and aplastic anemia and blood counts before, during, and after delivery. RESULTS: The 36 pregnancies resulted in 34 live births (one set of twins), 2 elective abortions, and 1 spontaneous abortion. Of the 36 pregnancies, 22 were uncomplicated and 14 involved medical complications. Seven pregnancies (19%) were complicated by relapse of aplastic anemia, and 5 patients without relapse (14%) needed transfusions during delivery. After delivery, 3 of the 7 patients who had relapse recovered spontaneously and 3 recovered after retreatment. One patient who did not respond to treatment died of aplastic anemia. A woman with aplastic anemia and paroxysmal nocturnal hemoglobinuria had a fatal cerebral thrombosis after delivery. Women with uneventful pregnancies had better prepregnancy remission status (8 complete and 11 partial remissions) and a higher median platelet count (146 x 10(9) cells/L) than did women with complicated pregnancies (2 complete remissions, 8 partial remissions, and 4 cases of paroxysmal nocturnal hemoglobinuria; median platelet count, 92 x 10(9) cells/L). CONCLUSIONS: Successful pregnancy with normal outcome is possible in women with aplastic anemia previously treated with immunosuppression. Complications appear to be more likely in patients with low platelet counts and paroxysmal nocturnal hemoglobinuria-associated aplastic anemia.
Adult↗