Discrimination of viable and non‐viable cells using propidium iodide in two color immunofluorescence

Sasaki, Dennis T.; Dumas, Stephanie E.; Engleman, Edgar G.

doi:10.1002/cyto.990080411

Cited by 176 publications

(79 citation statements)

References 18 publications

(5 reference statements)

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“…PI integrates with DNA in non-viable cells with compromised membranes and can be detected by flow cytometry. DCD was added to a suspension of non-fixed cells, 5 µL of DCD per 10 6 cells, immediately prior to FACS, performed as previously described [35] .…”

Section: Stimulation Experimentsmentioning

confidence: 99%

Potentially probiotic bacteria induce efficient maturation but differential cytokine production in human monocyte-derived dendritic cells

Latvala

Te²,

Veckman³

et al. 2008

WJG

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AIM:To analyze the ability of nine different potentially probiotic bacteria to induce maturation and cytokine production in human monocyte-derived dendritic cells (moDCs). METHODS: Cytokine production and maturation of moDCs in response to bacterial stimulation was analyzed with enzyme-linked immunosorbent assay (ELISA) and flow cytometric analysis (FACS), respectively. The kinetics of mRNA expression of cytokine genes was determined by Northern blotting. The involvement of different signaling pathways in cytokine gene expression was studied using specific pharmacological signaling inhibitors. RESULTS: All studied bacteria induced the maturation of moDCs in a dose-dependent manner. More detailed analysis with S. thermophilus THS, B. breve Bb99, and L. lactis subsp. cremoris ARH74 indicated that these bacteria induced the expression of moDC maturation markers HLA class Ⅱ and CD86 as efficiently as p a t h o g e n i c b a c t e r i a . H o w e ve r, t h e s e b a c t e r i a differed in their ability to induce moDC cytokine gene expression. S. thermophilus induced the expression of pro-inflammatory (TNF-α, IL-12, IL-6, and CCL20) and Th1 type (IL-12 and IFN-γ) cytokines, while B. breve and L. lactis were also potent inducers of antiinflammatory IL-10. Mitogen-activated protein kinase (MAPK) p38, phosphatidylinositol 3 (PI3) kinase, and nuclear factor-kappa B (NF-κB) signaling pathways were shown to be involved in bacteria-induced cytokine production. CONCLUSION: Our results indicate that potentially probiotic bacteria are able to induce moDC maturation, but their ability to induce cytokine gene expression varies significantly from one bacterial strain to another. Latvala S, Pietilä TE, Veckman V, Kekkonen RA, Tynkkynen S, Korpela R, Julkunen I. Potentially probiotic bacteria induce efficient maturation but differential cytokine production in human monocyte-derived dendritic cells.

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Section: Stimulation Experimentsmentioning

confidence: 99%

Potentially probiotic bacteria induce efficient maturation but differential cytokine production in human monocyte-derived dendritic cells

Latvala

Te²,

Veckman³

et al. 2008

WJG

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“…After expansion, cells were (Young et al, 1991 and greater than 98% by the propidium iodide [Calbiochem-Novabiochem Corporation, La Jolla, CA] exclusion technique (Sasaki et al, 1987). One hundred microliters of cell preparation (1 ϫ 10 5 cells) were stained with saturating concentrations of fluorescein isothiocyanate-(FITC), phycoerythrin-(PE), allophycocyanin (APC), or perdinin chlorophyll protein-(PerCP) conjugated CD3, CD4, CD8, CD11c, CD33, CD34, CD36, CD38, CD45, CD90, CD117, glycophorin-A, and HLA-II (DR), or isotype matched controls (Becton Dickinson, Inc. San Jose, CA).…”

Section: Flow Cytometrymentioning

confidence: 99%

Human reserve pluripotent mesenchymal stem cells are present in the connective tissues of skeletal muscle and dermis derived from fetal, adult, and geriatric donors

et al. 2001

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This study details the profile of 13 cell surface cluster differentiation markers on human reserve stem cells derived from connective tissues. Stem cells were isolated from the connective tissues of dermis and skeletal muscle derived from fetal, mature, and geriatric humans. An insulin/dexamethasone phenotypic bioassay was used to determine the identity of the stem cells from each population. All populations contained lineage-committed myogenic, adipogenic, chondrogenic, and osteogenic progenitor stem cells as well as lineageuncommitted pluripotent stem cells capable of forming muscle, adipocytes, cartilage, bone, fibroblasts, and endothelial cells. Flow cytometric analysis of adult stem cell populations revealed positive staining for CD34 and CD90 and negative staining for CD3, CD4, CD8, CD11c, CD33, CD36, CD38, CD45, CD117, Glycophorin-A, and HLA DR-II. Anat Rec 264: [51][52][53][54][55][56][57][58][59][60][61][62] 2001.

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“…Propidium iodide (PI) (Calbiochem, San Diego, CA) was added at a final concentration of 1 pg/ml, 30 min prior to analysis to allow dead cell discrimination (25,26).…”

Section: Immunofluorescent Stainingmentioning

confidence: 99%

Limited loss of nine tumor‐associated surface antigenic determinants after tryptic cell dissociation

et al. 1995

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Disodiumethylenediaminetetraacetic acid (EDTA) or trypsin/EDTA are Etequently used for the dispersion of monolayer cells into single cell suspensions allowing flow cytometric analysis of surface antigenic determinants. A disadvantage of EDTA is the slow action of this agent, whereas trypsin might affect the antigenic determinants under focus. We studied the possible deleterious effect of trypsin on three different ovarian carcinoma cell lines, COV413b, COv362.cl4, and NIH:OVCAR-3, on cell surface antigenic determinants by flow cytometry. Either EDTA or trypsin/EDTA was used for detachment and dissociation of monolayer ovarian cancer cell lines, followed by indirect immunofluorescence with a panel of monoclonal antibodies directed against nine different surhce antigenic determinants, including s k markers directed against widely distributed antigens. Compared to EDTA, trypsin/EDTA resulted in higher total cell yields and rapid detachment and dissociation into single cell suspensions with significantly lower amounts of dead cells detected by both trypan blue and propidium iodide (PI). Large differences in antigen expression were observed for the different cell lines.However, all antigenic determinants tested could still be detected after tryptic proteolysis. Three antigenic determinants were significantly decreased after trypsin/EDTA compared to EDTA detachment.CA 125 was decreased on CoV362.cl4 and NIH:OVCAR-3 cells, respectively. BMA 180 and ICAM-1 were decreased on COV413b cells. This cell linedependent decrease might be caused by differences in glycosylation. We conclude that trypsin/EDTA can be used for rapid monolayer cell detachment with high cell yields and limited loss of antigenic determinants tested. o 1995 Wiley-Liss, Inc.

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Discrimination of viable and non‐viable cells using propidium iodide in two color immunofluorescence

Cited by 176 publications

References 18 publications

Potentially probiotic bacteria induce efficient maturation but differential cytokine production in human monocyte-derived dendritic cells

Potentially probiotic bacteria induce efficient maturation but differential cytokine production in human monocyte-derived dendritic cells

Human reserve pluripotent mesenchymal stem cells are present in the connective tissues of skeletal muscle and dermis derived from fetal, adult, and geriatric donors

Limited loss of nine tumor‐associated surface antigenic determinants after tryptic cell dissociation

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