Light scatter analysis and sorting of cells activated in mixed leukocyte culture

MacDonald, H. Robson; Zaech, P.

doi:10.1002/cyto.990030112

Cited by 39 publications

(17 citation statements)

References 8 publications

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“…Samples were passed on a FACS I1 flow cytometer (Becton Dickinson, Sunnyvale, CA). Dead cells were gated out by a combination of forward and perpendicular light scatter [24]. Cytograms represent 5 x lo4 viable cells accumulated using logarithmic fluorescence amplification and displayed on an Ortho (Raritan, NJ) 2150 computer.…”

Section: Cytofluorometric Analysismentioning

confidence: 99%

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Gene transfer of the ly‐3 chain gene of the mouse cd8 molecular complex: co‐transfer with the ly‐2 polypeptide gene results in detectable cell surface expression of the ly‐3 antigenic determinants

et al. 1988

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The CD8 molecule is a glycoprotein expressed on a subset of mature T lymphocytes. It has been postulated to be a receptor for class I major histocompatibility complex molecules. In the mouse, CD8 is a heterodimer composed of Ly-2 and Ly-3 chains. We have isolated and analyzed cDNA and cosmid clones corresponding to the Ly-3 subunit. One of the isolated, cosmid clones was subsequently transfected, alone or in combination with the Ly-2 gene, into mouse Ltk- cells. Analysis of the Ly-2,3 molecules expressed at the surface of the double transfectants indicated that they are serologically and biochemically indistinguishable from their normal counterparts expressed on lymphoid cells. Ltk- cells transfected with the Ly-2 gene alone were shown to react with a subset of anti-CD8 monoclonal antibodies whereas Ly-3 transfectants did not stain with any of the anti-Ly-3 antibodies employed in this study. Since at least one of these antibodies (53-5.8) has been previously shown to recognize an epitope which is retained on the Ly-3 subunit after dissociation of the heterodimeric Ly-2,3 complex, these observations suggest that the expression of the Ly-2 polypeptide is required to permit the detectable cell surface expression of the antigenic determinants carried by the Ly-3 subunit.

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Section: Cytofluorometric Analysismentioning

confidence: 99%

“…Data are present as mean fluorescence intensity at equivalent linear amplifier gain (gain 1, 650 V) on a FACS I1 flow cytometer gated as described [24].…”

Section: Analysis Of the Cds Molecules Expressed By The M24 T Lymphomamentioning

confidence: 99%

Gene transfer of the ly‐3 chain gene of the mouse cd8 molecular complex: co‐transfer with the ly‐2 polypeptide gene results in detectable cell surface expression of the ly‐3 antigenic determinants

et al. 1988

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“…Due to increased size and granularity, blast transformation leads to increased forward and side scatter signals. This is well known and has been used to discriminate between activated and quiescent lymphocytes (16). An increased cell size should lead to an increase in both signal width and height, with FSC-A % FSC-W 3 FSC-H. During blast transformation, the cytoplasm and nucleus do not expand proportionately, and the refractive index and size of different cellular components contribute to the FSC signal in complex ways.…”

Section: Discussionmentioning

confidence: 99%

“…Activation of T cells is associated with an increase in the forward light scatter signal (FSC) (16). Although this parameter has been used to discriminate cell doublets, we explored its potential as a simple measure of proliferation for screening cytokine and drug effects on human T cells.…”

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Forward light scatter is a simple measure of T‐cell activation and proliferation but is not universally suited for doublet discrimination

2011

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Following activation by antigen, T cells enter the cell cycle in stages that can be defined with flow cytometric markers. We show that these markers include the increase of forward light scatter width (FSC-W) signal and the ratio of FSC area/peak. This change in light scatter precedes the first cell division and may reflect blast transformation. We show that the FSC-W parameter can be used, alone or in combination with other activation markers, to monitor the relative and absolute numbers of T cells responding to a proliferative stimulus. In contrast to dye dilution assays, the FSC-W method does not allow discrimination between consecutive cell divisions, but it has several advantages and could complement the dye dilution assay. Our findings also show that the routine elimination of doublets based on FSC signals may exclude proliferating T cells from the analysis. ' 2011 International Society for Advancement of Cytometry

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“…Samples were passed on a fluorescence-activated cell sorter (FACS 11, Becton Dickinson, Sunnyvale, CA) gated to exclude nonviable cells as described in detail previously [23].…”

Section: Immunofluorescent Staining and Flow Microfluorometrymentioning

confidence: 99%

Production and characterization of monoclonal anti‐Thy‐1 antibodies that stimulate lymphokine production by cytolytic T cell clones

et al. 1985

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In an effort to derive monoclonal antibodies (mAb) which can induce production of macrophage-activating factor (MAF) by cloned murine cytolytic T lymphocyte (CTL) lines, we have fused spleen cells from a rat immunized with a CTL clone with the nonsecreting mouse myeloma X63-Ag8.653. Three mAb (designated I-22, III-5 and V-8) were found to stimulate MAF production by the immunizing CTL clone and (with a single exception) two other unrelated CTL clones. However, none of these mAb inhibited the cytolytic activity of the clones. Immunoprecipitation studies indicated that the three mAb reacted primarily with a 25-30-kDa protein which could not be distinguished from that precipitated by either a reference anti-Thy-1.2 mAb or a polyclonal rabbit anti-Thy-1 antiserum. Moreover, competition binding experiments demonstrated that the three mAb competed with each other and with the reference anti-Thy-1.2 mAb. Flow cytofluorometric analysis of the strain distribution of the molecules defined by the mAb revealed that two of the antibodies (I-22 and III-5) were directed against nonpolymorphic determinants of Thy-1, whereas V-8 mAb reacted only with Thy-1.2+ lymphocytes. One of the mAb (III-5) was also able to stimulate proliferation and interleukin 2 secretion by normal splenic T cells. Since mAb directed against a number of other surface structures on CTL clones did not stimulate MAF production, it thus appears that Thy-1 (or molecules associated with Thy-1) may play a functional role in T lymphocyte triggering.

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Light scatter analysis and sorting of cells activated in mixed leukocyte culture

Cited by 39 publications

References 8 publications

Gene transfer of the ly‐3 chain gene of the mouse cd8 molecular complex: co‐transfer with the ly‐2 polypeptide gene results in detectable cell surface expression of the ly‐3 antigenic determinants

Gene transfer of the ly‐3 chain gene of the mouse cd8 molecular complex: co‐transfer with the ly‐2 polypeptide gene results in detectable cell surface expression of the ly‐3 antigenic determinants

Forward light scatter is a simple measure of T‐cell activation and proliferation but is not universally suited for doublet discrimination

Production and characterization of monoclonal anti‐Thy‐1 antibodies that stimulate lymphokine production by cytolytic T cell clones

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