Flow Cytometry

We have analyzed cell cycle parameters for the Aedes albopictus C7-10 mosquito cell line, which has been systematically developed for somatic cell genetics, expression of transfected genes, and synthesis of hormone-inducible proteins. In rapidly cycling cells, we measured a generation time of 10-12 h. The duration of mitosis (M) was < or = 1 h, and the DNA synthesis phase (S) required 6 h. Unlike Drosophila melanogaster Kc cells, in which the G2 gap is substantially longer than G1, in C7-10 cells G1 and G2 each lasted approximately 2 h. In these cells, the duration of both S and G2 was independent of the population doubling time, and the increase in population doubling time as cells approached confluency was due to prolongation of G1. When treated with the insect steroid hormone, 20-hydroxyecdysone, C7-10 mosquito cells complete the cycle in progress before undergoing a reversible arrest.

Objective. Blood platelets represent a link between hemostasis, inflammation, and tissue repair. Their role in immune responses and inflammation mainly involves many molecules, among which Toll-like receptor, major histocompatibility complex class I, CD40 and CD154/CD40 ligand (CD40L). As platelets are the major purveyor of soluble CD40L (sCD40L), we sought to determine their involvement in CD40/CD40L-dependent immune responses and to understand the interactions between platelets and peripheral B lymphocytes. Materials and Methods. We examined the capacity of platelets to bind nonstimulated B cells, and phenotypic changes by flow cytometry and confocal scanning laser microscopy. Modulation of cytokines/chemokines and total levels of immunoglobulin (Ig) A, IgG, IgM, and IgG subclasses in supernatants of coculture, platelets, and B lymphocytes was performed by sandwich enzyme-linked immunosorbent assay and differential production of cytokine mRNA as determined by reverse transcriptase polymerase chain reaction. Results. In coculture, platelets and B lymphocytes were mutually activated, as demonstrated by the increased expression of platelet CD62p and B-cell CD86. Platelet/B-cell interactions were accompanied by changes in membrane expression of CD40 and CD40L by both platelets and B lymphocytes. IL12p70 and IL8 gene transcription were significantly reduced, which was attributable to B cells. Conversely, there was a significant, platelet-dependent reduction of sCD40L and RANTES mRNA expression. After a 3-day incubation with platelets, differentiated B cells increased their in vitro production of IgG1, IgG2, and IgG3, but not IgG4, IgA, or IgM. Conclusion. These data emphasize the potentially important role of platelets in the adaptive immune response. Platelets have an immunoregulatory role that might be applied clinically in multitransfused patients (e.g., hematopoietic stem cell transplantation). Ó

The 19 chromosomal pairs of the swine karyotype are resolved into 18 peaks denoted A to Q and Y by dual-beam flow cytometry. The chromosomal content of six peaks has previously been determined by analyzing male/female differences, karyotypes of animals carrying translocations, and PCR studies of genes with known assignments. For the remaining chromosomes, putative assignments to flow peaks were deduced from comparison of DNA contents, determined by flow cytometry, and chromosomal size. We present here the complete characterization of the pig bivariate flow karyotype using the PARM-PCR technique combined with fluorescence in situ hybridization. Chromosome-specific probes were generated by PCR amplification of 300 sorted chromosomes with primers under nonspecific conditions and used to paint chromosomes by FISH. The chromosomal content of each peak was identified: peaks A (chromosome 1), B (13), C (6), D (2), E (14), F (3), G (7), H (9,4,X), H1 (9), I (15), J (8), K (5), L (10), M (12), N (16), O (11), P (17), Q (18), Y (Y). We were able to characterize perfectly the pig bivariate flow karyotype. Such techniques could be applied to any other species.

Overexpression of Bcl-2 protein has been observed in more than 80% of B-cell lymphomas, including diffuse large cell lymphoma (DLCL), the most common subtype of non-Hodgkin's lymphoma. We have previously employed the natural product (À)-gossypol to test its therapeutic potential as a small-molecule inhibitor of Bcl-2 for the treatment of B-cell lymphomas. Experimental Design: Recently, we have used a structure-based strategy to design a new class of potent small-molecule inhibitor acting on Bcl-2. One such lead compound is the benzenesulfonyl derivative TW-37, which was designed to target the BH3-binding groove in Bcl-2 where proapoptotic Bcl-2 proteins, such as Bak, Bax, Bid, and Bim bind. Results: In our fluorescence polarization^based binding assays using recombinant Bcl-2, Bcl-X L , and Mcl-1proteins,TW-37 binds to Bcl-2, Bcl-X L , and Mcl-1with K i values of 290, 1,110 and 260 nmol/L, respectively. Hence,TW-37 is a potent inhibitor of Bcl-2 and has >3-fold selectivity over Bcl-X L. In vitro,TW-37 showed significant antiproliferative effect in a de novo chemoresistant WSU-DLCL 2 lymphoma cell line and primary cells obtained from a lymphoma patient with no effect on normal peripheral blood lymphocytes. Coimmunoprecipitation experiments showed that TW-37 disrupted heterodimer formation between Bax or truncated-Bid and antiapoptotic proteins in the order Mcl-1 > Bcl-2 >> Bcl-X L. As expected, TW-37 caused apoptotic death. Pre-exposure of lymphoma cells toTW-37 significantly enhanced the killing effect of cyclophosphamide-doxorubicin-vincristine-prednisone (CHOP) regimen. The maximum tolerated dose of TW-37 in severe combined immunodeficient (SCID) mice was 40 mg/kg for three i.v. injections when given alone and 20 mg/kg, Â3 when given in combination with CHOP. UsingWSU-DLCL 2-SCID mouse xenograft model, the addition of TW-37 to CHOP resulted in more complete tumor inhibition compared with either CHOP orTW-37 alone. Conclusions: We conclude that the administration of TW-37, as a potent Bcl-2 and Mcl-1inhibitor, to standard chemotherapy may prove an effective strategy in the treatment of B-celllymphoma.

G ROWING numbers of patients on renal transplantation waiting lists have HLA-specific alloantibodies, factors that are responsible for positive crossmatch and/or high panel-reactive antibody (PRA) levels. Many clinical studies have focused on suppressing or eliminating anti-HLA antibodies through the use of immunosuppressive drugs, immunoadsorption, or intravenous immunoglobulin. The results of these trials have been inconsistent and disappointing, and there have also been problems with side effects and cost. There is a need for safer and more effective therapy in the treatment of HLA sensitization.

Background: Until now, the simultaneous analysis of several parameters during apoptosis, including DNA content and mitochondrial membrane potential (DW), has not been possible because of the spectral characteristics of the commonly used dyes. Using polychromatic flow cytometry based upon multiple laser and UV lamp excitation, we have characterized cells with different DW during apoptosis. Methods: U937 cells were treated with the flavonoid quercetin (Qu) and stained with JC-1 to detect DW, propidium iodide (PI) for cell viability, Hoechst 33342 for DNA content, Annexin V conjugated with Alexa Fluor-647 for detection of phosphatidilserine (PS) exposure, marker of early apoptosis, or Mitotracker Deep Red for the determination of mitochondrial mass. Results: Treatment with Qu provoked the onset of three cell populations with different DW: (1) healthy cells, with normal DW, DNA content and physical parameters, high mitochondrial mass, PI-and Annexin V-negative; (2) cells with intermediate DW and normal DNA content, but with physical parameters typical of apoptotic cells and low mitochondrial mass; most of them were PI1 and Annexin V1; (3) cells with collapsed DW that had low mitochondrial mass and were Annexin-V1, PI1; half of them showed diminished DNA content. Similar results, i.e. the presence of cells with intermediate DW, were observed in other models of apoptosis. Conclusions: During Qu-induced apoptosis, loss of DW, PS exposure, and decrease of mitochondrial mass are early events that precede permeability to PI and loss of DNA. Populations of cells with different DW, as revealed by flow cytometry after JC-1 staining, differed also for other parameters associated to apoptosis. Thus, the simultaneous analysis of several parameters by polychromatic flow cytometry permits a better identification of many stages of cell death, and, more in general, allows to evaluate the eventual heterogenic sensibility of the population under study to a given compound. q 2005 International Society for Analytical Cytology