Abstract: We have used fluorescence-activated cell sorting with class-specific antisera to isolate spontaneous variants in the expression of immunoglobulin heavy chain class from the mouse myeloma cell line X63 (IgGI, kappa). In the wild-type cell population, only one type of variants was found, namely, cells expressing IgG2b. From an IgG2b variant clone we isolated secondary variants that had either reverted to IgGI expression or expressed IgG2a or IgG2a and IgG2b concomitantly. The variant heavy chains are of normal size. The variant immunoglobulins were characterized serologically, and all of them still expressed the wild-type idiotype. Wild-type and variant cell populations were screened for heavy chain class-switch variants by fluorescence microscopy. A variety of switch variants was found in addition to the ones isolated by cell sorting, and a clear pattern of class switching (gamma 1 leads to gamma 2b leads to gamma 2a leads to alpha) with frequent reversion emerges from this analysis. Cells expressing new heavy chain classes occurred at frequencies of about 10(-7)-10(-6)/cell per generation, whereas revertants were as frequent as 10(-6)-10(-5)/cell per generation.

Abstract: A procedure has been investigated for sorting viable cells according to their DNA content. Cells are stained with the U.V. activated fluorochromes 4'6-diamidino-2-phenylindole (DAPI), Hoechst 33258 or Hoechst 33342, and sorted with a Fluorescence Activated Cell Sorter. Hoechst 33342 is a suitable vital stain for a variety of cell types. Hoechst 33258 and DAPI, however, are quantitative vital stains for CHO cells only. Cloning efficiency is unaffected by the sorting procedure, and these stains are not mutagenic at concentrations suitable for vital staining. Potential applications of this procedure to cell biology are discussed.

Abstract: Cell sorting and tritiated thymidine autoradiography were used to define the distribution of S phase cells in flow cytometric DNA histograms obtained from exponential mouse lymphoma cells (L5178Y). The numbers of labeled S phase cells, autoradiographically determined from cells, autoradiographically determined from cells sorted at 2-channel intervals in the G/sub 1//early S and late S/G/sub 2/M regions of the histogram, were compared with the numbers of computed S phase cells in comparable 2-channel intervals as predicted by several computer algorithms used to extract cell cycle phase distributions from DNA histograms. Polynomial and multirectangle algorithms gave computed estimates of total %S in close agreement with the tritiated thymidine labeling index for the cell population, while multi-Gaussian algorithms underestimated %S. Interval autoradiographic and algorithm studies confirmed these results in that no significant differences were found between the autoradiographic S phase distribution and S phase distributions calculated by the polynomial and multirectangle models. However, S phase cells were significantly underestimated by G/sub 1//early S by a constrained multi-Gaussian model and in both G/sub 1//early S and late S/G/sub 2/ by an unconstrained multi-Gaussian model. For the particular cell line (L5178Y), staining protocol (mithramycin following ethanol fixation) and instrumentation (Coulter TPS-2 cell sorter) usedmore » in this study, close agreement between computed %S and tritiated thymidine labeling index was found to be a reliable indicator of an algorithm's success in resolving S phase cells in the G/sub 1//S and S/G/sub 2/ transition regions of the DNA histograms.« less

Abstract: Mammalian monoclonal antibodies specific for an antigen diagnostic for thymocytes, normal peripheral T cells and some null cells. The antibodies which distinguish among subpopulations of T cells, find use in assays, cell sorting, and immunosupression.

Abstract: Light-activated cell sorting although capable of highly specific selection of subpopulations of cells is rate-limited. The method as it now exists is insufficient to supply an adequate number of stem cells for bone marrow transplantation. However, other separation methods using electrophoresis, density gradients, and density centrifugation coupled with cell sorting can improve the yield for clinical utilization.

Abstract: Liver cells were isolated by a collagenase perfusion technique. A fraction of the cell suspension containing mostly parenchymal cells was obtained by slow speed centrifugation. Separated, intact cells were stained with mitramycin for flow cytometry. A brief pre-treatment with toluene was found necessary for rapid and uniform staining. Isolated nuclei were prepared by a combination of detergent and pepsin treatments and stained with ethidium bromide.

Abstract: Conditioned media prepared from human lung, placenta, peripheral leukocytes, cultured human pancreatic carcinoma cells, and cultured cervical carcinoma cells exhibit a common pattern of two distinct types of colony-stimulating factors (CSF) separable by isoelectrofocusing. Type I and type II CSF differ in MW, isoelectric point, CFU-C specificity, and the morphologic type of colonies they stimulate. Type I CSF exhibits higher activity in mouse than in human marrow while type II is more active in human marrow. Type I and II CSF from cultured human pancreatic carcinoma have been purified, type I to apparent homogeneity, and antibody has been prepared against them in rabbits. We have utilized purified CSF and anti-CSF antibodies to label CFU-C fluorescently for the purpose of cell sorting via flow photometry. Human bone marrow cells preincubated with CSF and then treated first with anti-CSF antibody then fluorescein-labeled goat antirabbit globulin retain their ability to grow and form aggregates in the presence of additional CSF. Colonies thus formed exhibit fluorescence, the intensity of which diminishes with increase in aggregate size. These observations provide new insight into the biology of CFU-C and suggest the following: (1) Incubation of marrow cells with CSF for 2 h results in binding of CSF or an antigenic component of CSF to membranes of CFU-C. (2) Bound CSF-anti CSF complex remains on CFU-C membranes through at least 5-6 cell divisions. (3) The approach described offers great potential for the preparation of highly purified CFU-C populations by fluorescence cell sorting.

Abstract: Cell sorting has been used as an approach to describing surface markers and functional receptors of hemopoietic stem cells and progenitors. Significant enrichment of CFU-S was obtained with fluorescent cell sorting of human antihuman sperm-labelled bone marrow cells; significant enrichment of human CFU-C was obtained by cell sorting of hybridoma (KGP1)-labelled human bone marrow cells. Significant changes in human bone marrow CFU-C, but not CFU-E were observed by fluorescence polarization after exposure to a putative stimulator of CFU-C. A description of preliminary investigations of a hybrid clone from Dexter culture cells x Chinese hamster ovary and fluorescence polarization measurements after exposure to purified mouse-lung CSF are also presented.

Abstract: Antigen binding was used as a probe in the definition of functional B cell heterogeneity. Unprimed, anti-Thy 1 and complement-treated spleen cells were stained with fluorescent trinitrophenylated bovine serum albumin (FL-TNP-BSA). These cells were sorted, fluorescence negative from fluorescence positive, by using a multiparameter cell sorter and assayed for precursor frequency in antibody responses to TNP by limiting dilution analysis. The cells that bound FL-TNP-BSA were demonstrated to be enriched for antibody-forming precursors to the antigens TNP-lipopolysaccharide (LPS), TNP-sheep red blood cells (SRBC), and TNP- or DNP-Ficoll, whereas the fluorescence negative cells were depleted for these responses. B cells that bound FL-TNP-BSA were then sorted into populations that bound a moderate or high amount of FL-TNP-BSA. The B cells responsive to TNP-LPS and TNP-SRBC were present in both the moderate and high binding populations. In contrast, the B cells responsive to TNP- or DNP-Ficoll were present only in the cells that bound a moderate amount of FL-TNP-BSA. These experiments suggest that there is a population of B cells in adult mouse spleen that binds large amounts of antigen, and that can respond to antigen carried on LPS or SRBC but not carried on Ficoll.

Abstract: A reversible growth arrest of simian virus 40-transformed human fibroblasts has been produced by replacement of methionine in the growth medium by its immediate metabolic precursor, homocysteine, Although these arrested cells exhibit a greatly reduced cloning efficiency when plated in methionine-supplemented medium, they resume rapid proliferation without a lag when subconfluent cells are refed with methionine-supplemented medium. This growth arrest is accompanied by a reduction in the percentage of mitotic cells in the cell population. Furthermore, data obtained using fluorescence-activated cell sorting techniques indicate that the cells are arrested i the S and G2 phases of the cell cycle. This is in contrast to a G1-phase accumulation of cells, which occurs only in methionine-supplemented medium at very high densities and which is similar to the G1 block seen in cultures of normal fibroblasts at high density. The apparent relationship between specific events in the DNA-synthetic and premitotic phase of the cell cycle and methionine dependence in these transformed cultures is discussed.

Abstract: A hybridoma-secreting monoclonal antibody was produced from the spleen cells of a mouse immunized with human thymocytes. This hybridoma antibody, termed OKT5, was reactive by indirect immunofluorescence with 80% of human thymocytes but only 20% of peripheral blood T cells. Moreover, OKT5 was unreactive with normal B cells, null cells, and macrophages at any dilution tested. A similar pattern of reactivity was seen with an equine antiserum to human thymocytes termed anti-TH2. Fluorescence-activated cell sorting demonstrated that the OKT5 antibody reactivity on peripheral T cells was restricted to the majority of the previously defined TH2+ subpopulation. In functional studies, the OKT5+ subset, like the TH2+ subset, proliferated well to the mitogen Con A and to alloantigens, and contained cytotoxic effector cells after sensitization in MLC, and suppressor effector cells after activation with Con A. In addition, like the TH2+ T cell, the OKT+ T cell was virtually unresponsive to soluble antigen. Thus, the OKT5 monoclonal antibody is reactive with the cytotoxic/suppressor T cell subset. OKT5 should provide an important probe to assess the status of suppressor cells in human disease.