Enumeration of cytoplasmic μ immunoglobulin positive acute lymphoblastic leukemia cells by flow cytometry: Comparison with fluorescence microscopy

Zipf, Theodore F.; Bryant, Laurette D.; Koskowich, G. N.; MacGregor, S. E.; Chin, Laura D.; Johnson, Howard M.

doi:10.1002/cyto.990050609

Cited by 14 publications

(2 citation statements)

References 26 publications

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“…NALM-6, a pre-B ALL line, expresses intracytoplasmic p but does not display any surface IgM (8). Flow cytometric evaluation of intracellular staining of p in this cell line has been described before by using ethanol fixation to permeabilize the cells (24). We have demonstrated here that paraformaldehyde fixation, which is preferable to alcohol fixation because it minimizes cell loss and creation of debris (3), can be used with very good results.…”

Section: Application 2: Detection Ofmentioning

confidence: 73%

A gentle fixation and permeabilization method for combined cell surface and intracellular staining with improved precision in DNA quantification

1991

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A method was developed for gentle fixation of mammalian cells and permeabilization of their membranes. The method is useful for staining of intracellular antigens or quantification of DNA content simultaneously with cell surface staining. Cells are treated for 1 h at 4°C with 0.25% buffered paraformaldehyde then for 15 min at 37°C with 0.2% Tween 20 detergent in PBS. The procedure permits excellent staining of intracellular proteins, very low coefficients of variation (CV) on the G,G,-peak of DNA distributions, and preservation of the integrity of cell surface antigens. The low vs. 90" angle light scatter profile of cell clusters is maintained thereby allowing discrimination of different cell populations including human peripheral blood lymphocytes and monocytes for gating and analytic purposes. The method was successfully used on a variety of other cell types, including human thymocytes, murine thymocytes and spleen cells, and several leukemic cell lines. Dual-color surface antigen staining combined with DNA staining with 7-amino-actinomycin D (7-AAD) on peripheral blood mononuclear cells (PBMC) cultured with tetanus toxoid allowed the determination of the cell subset that was preferentially stimulated. Staining for internal antigens was done on CCRF-CEM for expression of C D~E and on NALM-6 for expression of p. The technique we developed gave bright and specific staining of internal antigens in the examples presented here. It is particularly suited for correlations of internal antigen staining with DNA staining and/or surface immunofluorescence.

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Section: Application 2: Detection Ofmentioning

confidence: 73%

A gentle fixation and permeabilization method for combined cell surface and intracellular staining with improved precision in DNA quantification

1991

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“…Evaluation of this marker in CD8+ T cells in normal and HIV + individuals demonstrated an elevated proportion of cells from HIV + individuals ex-pressing the TIA-1 antigen. A variety of other studies have attempted to evaluate intracellular antigens in T lymphocytes and many have focused on detection of cytokines, including interleukin-2, interleukin-4, gamma interferon, and tumor necrosis factor (2,15, 18,311, or on B cell evaluation for cytoplasmic immunoglobulin (32). These reports point the way for future studies of whole blood samples for the detection of cytoplasmically expressed functional molecules.…”

Section: Discussionmentioning

confidence: 99%

Whole blood method for simultaneous detection of surface and cytoplasmic antigens by flow cytometry

Landay

Jennings

Forman³

et al. 1993

Cytometry

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Dual parameter analysis of surface antigens in flow cytometry has become a standard method for detection of cell subsets. However, few methods have been described for the extension of multiparameter analyses to include cytoplasmic or intracellular antigens. Here we describe a simple and reproducible method for simultaneous detection of surface and intracellular antigens by flow cytometry in lysed whole blood samples. This method employs the use of digitonin, a mild glycoside detergent, and formaldehyde for permeabilization and fixation. Red blood cells are lysed with 2% acetic acid. Preparation of samples in this manner resulted in altered light scatter characteristics relative to unpermeabilized samples; however, gating issues were overcome using a combination of scatter vs. fluorescence gating. Quantitation of CD3+/CD4+ and CD3+/CDS+ cells using this method was equivalent to counts obtained with the reference method using a commercially available lysis procedure and fluorescence vs. scatter gating. The effectiveness of the permeabilization process was assessed using a monoclonal antibody designated TIA-1, which is specific for a cytoplasmic antigen associated with cytotoxic granules predominantly found in CDS+ cells. The method effectively quantitated TIA-1 positive cells and demonstrated the specificity of the reagent for a subpopulation of CD8+ lymphocytes. Using this simplified procedure for simultaneous identification of surface and cytoplasmic antigens could help in studies of cell activation, proliferation, and other functional characteristics of the immune system.

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A flow‐cytometric analysis of hybridoma growth and monoclonal antibody production

Dalili¹,

Ollis²

1990

Biotech & Bioengineering

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A flow cytometric kinetic study of hybridoma growth and monoclonal antibody production is presented, along with the influence of glutamine on intracellular responses such as (relative) cell size, and cell RNA and total protein content. Specific findings are: (1) RNA content remained constant throughout the growth phase, then fell drastically as the cells entered the stationary phase. Also, in stationary phase, RNA content of antibody-producing cells was higher than for those not secreting antibody. (2) The cell size was constant and maximal throughout exponential phase, and diminished monotonically during later stages. (3) Average protein and antibody cellular content declined dramatically upon glutamine exhaustion. Thus, relative RNA levels and cell size provided quantitative determinants of both cell growth state and antibody secretion conditions. These results encourage consideration of structured kinetic studies which recognize the quality of the biophase.

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Enumeration of cytoplasmic μ immunoglobulin positive acute lymphoblastic leukemia cells by flow cytometry: Comparison with fluorescence microscopy

Cited by 14 publications

References 26 publications

A gentle fixation and permeabilization method for combined cell surface and intracellular staining with improved precision in DNA quantification

A gentle fixation and permeabilization method for combined cell surface and intracellular staining with improved precision in DNA quantification

Whole blood method for simultaneous detection of surface and cytoplasmic antigens by flow cytometry

A flow‐cytometric analysis of hybridoma growth and monoclonal antibody production

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