Dead cell discrimination with 7‐amino‐actinomcin D in combination with dual color immunofluorescence in single laser flow cytometry

Schmid, Ingrid; Krall, Wanda J.; Uíttenbogaart, Christel H.; Braun, Jonathan; Giorgi, Janis V.

doi:10.1002/cyto.990130216

Cited by 419 publications

(262 citation statements)

References 11 publications

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“…Early stage apoptosis was measured using annexin V-Fluos (Roche Molecular Biochemicals) in conjunction with 7-AAD (Sigma) to distinguish early apoptosis, late apoptosis, and necrosis [39,40]. Cells were plated in T-75 flasks in medium containing 10% FBS.…”

Section: Apoptosismentioning

confidence: 99%

“…Viable cells were electronically gated on forward and side scatter parameters. In addition, viable cells were gated on their ability to exclude the dye 7-AAD [39,40].…”

Section: Apoptosismentioning

confidence: 99%

“…When coupled with 7-AAD, the Annexin V method differentiates between apoptotic and necrotic cells [39,40]. Apoptosis was assessed in cells overexpressing between 55-fold and 75-fold higher PTHrP levels than parental cells.…”

Section: Pthrp Increases Lovo Cell Survivalmentioning

confidence: 99%

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Increased cell survival, migration, invasion, and Akt expression in PTHrP-overexpressing LoVo colon cancer cell lines

Shen¹,

Mula²,

Evers³

et al. 2007

Regulatory Peptides

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Parathyroid hormone-related protein (PTHrP) has been localized in human colon cancer tissue and cell lines. We have previously shown that PTHrP increases colon cancer cell proliferation, extracellular matrix adhesion, and cell-surface integrin α6β4 expression. Since cancer cell migration, invasion, and survival are crucial components of metastasis, and colon cancer has a high metastatic potential, in this study we used the human colon cancer cell line LoVo as a model system to study the effects of PTHrP on these parameters. PTHrP expression was modulated by stable transfection with a construct expressing PTHrP (−36 to +139). We report that PTHrP increases cell migration, invasion, and survival. PTHrP altered cell morphology, with PTHrP-overexpressing cells exhibiting increased spreading and several long protrusions. PTHrP also increased the steady-state mRNA levels of the integrin α6 and β4 subunits, indicating a direct and/or indirect effect of PTHrP on the transcriptional and/or posttranscriptional regulation of integrin α6 and β4 expression. Integrin α6β4 activates the phosphoinositol 3-kinase (PI3-K)/Akt pathway, leading to glycogen synthase kinase-3 (GSK-3) deactivation. PTHrP overexpression also led to an increase in active Akt and inactive GSK-3 levels, indicating that the PTHrP-mediated upregulation of integrin α6β4 expression may activate the PI3-K pathway, resulting in increased cell survival, migration and invasion.

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Section: Apoptosismentioning

confidence: 99%

“…Viable cells were electronically gated on forward and side scatter parameters. In addition, viable cells were gated on their ability to exclude the dye 7-AAD [39,40].…”

Section: Apoptosismentioning

confidence: 99%

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Increased cell survival, migration, invasion, and Akt expression in PTHrP-overexpressing LoVo colon cancer cell lines

Shen¹,

Mula²,

Evers³

et al. 2007

Regulatory Peptides

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“…Surface and intracellular immunophenotyping of purified human thymocytes and PBMC with directly conjugated Abs was performed as previously described (48,49). Briefly, for intracellular staining, cells were surface immunophenotyped, fixed in 1% paraformaldehyde, and subsequently permeabilized in 0.2% Tween 20 for 15 min at 37°C.…”

Section: Immunofluorescent Intracellular Staining and Flow Cytometrymentioning

confidence: 99%

TCRγδ+ and CD161+ Thymocytes Express HIV-1 in the SCID-hu Mouse, Potentially Contributing to Immune Dysfunction in HIV Infection

Gurney

Yang

Wilson

et al. 2002

The Journal of Immunology

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The vast diversity of the T cell repertoire renders the adaptive immune response capable of recognizing a broad spectrum of potential antigenic peptides. However, certain T cell rearrangements are conserved for recognition of specific pathogens, as is the case for TCRγδ cells. In addition, an immunoregulatory class of T cells expressing the NK receptor protein 1A (CD161) responds to nonpeptide Ags presented on the MHC-like CD1d molecule. The effect of HIV-1 infection on these specialized T cells in the thymus was studied using the SCID-hu mouse model. We were able to identify CD161-expressing CD3+ cells but not the CD1d-restricted invariant Vα24/Vβ11/CD161+ NK T cells in the thymus. A subset of TCRγδ cells and CD161-expressing thymocytes express CD4, CXCR4, and CCR5 during development in the thymus and are susceptible to HIV-1 infection. TCRγδ thymocytes were productively infectable by both X4 and R5 virus, and thymic HIV-1 infection induced depletion of CD4+ TCRγδ cells. Similarly, CD4+CD161+ thymocytes were depleted by thymic HIV-1 infection, leading to enrichment of CD4−CD161+ thymocytes. Furthermore, compared with the general CD4-negative thymocyte population, CD4−CD161+ NK T thymocytes exhibited as much as a 27-fold lower frequency of virus-expressing cells. We conclude that HIV-1 infection and/or disruption of cells important in both innate and acquired immunity may contribute to the overall immune dysfunction seen in HIV-1 disease.

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“…Since many investigators became interested in multiparameter analysis, PI has also been used in combination with antibody labeling (16). Because of the large spectral overlap of PE and PI fluorescence, simultaneous measurement of FITC, PE, and PI has been considered to be difficult (12,13,15). Recent publications showed the use of PI for this purpose (3,4), however, compensation difficulties still remain.…”

mentioning

confidence: 99%

TO‐PRO‐3 iodide: A novel HeNe laser‐excitable DNA stain as an alternative for propidium iodide in multiparameter flow cytometry

1994

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A new red emitting fluorophore, TO-PRO-3 iodide (TP3), which is best excited by an HeNe laser (633 nm), has been compared with propidium iodide (PI) for measuring relative DNA content. TP3, which has a peak absorbance at 642 nm and emission at 661 nm, has been tested on peripheral blood lymphocytes (PBL) and keratinocytes in a two-laser system. As an example, we present a three-color flow cytometric application utilizing TP3 in combination with fluorescein-isothiocyanate (FITC) and phycoerythrin (PE) conjugated to monoclonal antibodies in this paper.A subequilibrium concentration of 1 pM TP3, most preferably used in combination with RNase treatment, proved to be a powerful alternative for DNA amount determination. In humanand mouse-Balb/MK-keratinocyte populations with different S-phase fractions, PI and TP3 showed a good correlation.Finally, in the triple labelling experiment we clearly demonstrated that TP3 is readily applied to the analysis of binding of two antibodies and relative DNA content simultaneously. 0 1994 Wiley-Liss, Inc.

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Dead cell discrimination with 7‐amino‐actinomcin D in combination with dual color immunofluorescence in single laser flow cytometry

Cited by 419 publications

References 11 publications

Increased cell survival, migration, invasion, and Akt expression in PTHrP-overexpressing LoVo colon cancer cell lines

Increased cell survival, migration, invasion, and Akt expression in PTHrP-overexpressing LoVo colon cancer cell lines

TCRγδ+ and CD161+ Thymocytes Express HIV-1 in the SCID-hu Mouse, Potentially Contributing to Immune Dysfunction in HIV Infection

TO‐PRO‐3 iodide: A novel HeNe laser‐excitable DNA stain as an alternative for propidium iodide in multiparameter flow cytometry

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