An improved fluorescence assay for the determination of lymphocyte-mediated cytotoxicity using flow cytometry

Papadopoulos, Nikolaos G.; Dedoussis, George; Spanakos, Gregory; Gritzapis, Angelos D.; Baxevanis, Constantin N.; Papamichail, Michael

doi:10.1016/0022-1759(94)90147-3

Cited by 258 publications

(162 citation statements)

References 25 publications

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“…The measurement of NK cytotoxic activity using this EGFP-flow cytometric assay not only showed a high correlation with the standard 51 Cr release assay, but also enabled to cut the time period required for assay, as previously reported (13). Several publications have highlighted a significant correlation between 51 Cr release assays, fluorescent dye release assay, and flow cytometric assay (13,15,17,19,24). Our development and optimization of the test demonstrate several interesting and crucial features of these analytical conditions: (i) A reduced volume of blood (i.e., 4 ml), for 3-6 assays depending on the E/T ratio 5:1 or 10:1 used and depending on the percentage of NK cells in the blood of each volunteer (4-37%).…”

Section: Effect Of Feeding On Nk Functions In Healthy Elderly Peoplesupporting

confidence: 65%

“…(iii) Concerning the effector-target cells incubation time, 1 h period shows significant results and 4 h appears as optimal. To correctly evaluate the ability of NK cells to attack target cells, it is necessary to set NK number in the PBMC population, contrary to other assays that set total PBMC number (13,17,24). Finally, flow cytometric assay has the advantage of simultaneously measuring three different parameters: percentage of NK cells, absolute number of NK cells, and fluorescence intensity of labeled dead cells, thereby allowing a detailed interpretation of NK cytotoxic functions.…”

Section: Effect Of Feeding On Nk Functions In Healthy Elderly Peoplementioning

confidence: 99%

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Implementation of the EGFP‐K562 flow cytometric NK test: Determination of NK cytotoxic activity in healthy elderly volunteers before and after feeding

Allegra¹,

Deleine²,

Michael-Jubely³

et al. 2006

Cytometry Pt A

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Background: Natural Killer (NK) cells are key actors of innate immunity that supervise the organism's cells, and fight against viral infections and cancer development through their cytotoxic activity. This cytotoxic activity is modulated by cytokines and hormones and could be influenced by physiological or pathological conditions. New techniques for measuring NK cytotoxic activity by flow-cytometry have recently been developed, and they correlated strongly with the standard chromium ( 51 Cr) release assay. Our aim was to implement a previously published enhanced green fluorescent protein (EGFP)-K562 flow cytometric method and use it to evaluate NK cytotoxic activity under different nutritional conditions. Methods: NK effector cells were isolated from peripheral blood mononuclear cells, and a K562 cell line stably transfected by EGFP was used as target cells. Different ana-

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Section: Effect Of Feeding On Nk Functions In Healthy Elderly Peoplesupporting

confidence: 65%

Section: Effect Of Feeding On Nk Functions In Healthy Elderly Peoplementioning

confidence: 99%

Implementation of the EGFP‐K562 flow cytometric NK test: Determination of NK cytotoxic activity in healthy elderly volunteers before and after feeding

Allegra¹,

Deleine²,

Michael-Jubely³

et al. 2006

Cytometry Pt A

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“…The Molecular Probes live/dead assay was also utilized, following the manufacturer's recommendation, on similarly treated cells. 27 In this assay, cell viability is assessed by intracellular esterase activity (cleavage of the AM-ester group) to label live cells. Dead cells are identified by the failure of the membrane barrier to exclude the DNA-labeling dye, ethidium homodimers ( Fig.…”

Section: Cell Viability After Vacuum Treatmentmentioning

confidence: 99%

A Rapid Seeding Technique for the Assembly of Large Cell/Scaffold Composite Constructs

Solchaga¹,

Tognana²,

Penick³

et al. 2006

Tissue Engineering

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These studies address critical technical issues involved in creating human mesenchymal stem cell (hMSC)/scaffold implants for cartilage repair. These issues include obtaining a high cell density and uniform spatial cell distribution within the scaffold, factors that are critical in the initiation and homogeneity of chondrogenic differentiation. For any given scaffold, the initial seeding influences cell density, retention, and spatial distribution within the scaffold, which eventually will affect the function of the construct. Here, we discuss the development of a vacuum-aided seeding technique for HYAFF ® -11 sponges which we compared to passive infiltration. Our results show that, under the conditions tested, hMSCs were quantitatively and homogeneously loaded into the scaffolds with 90+% retention rates after 24 h in perfusion culture with no negative effect on cell viability or chondrogenic potential. The retention rates of the vacuum-seeded constructs were at least 2 times greater than those of passively seeded constructs at 72 h. Histomorphometric analysis revealed that the core of the vacuum-seeded constructs contained 240% more cells than the core of passively infiltrated scaffolds. The vacuum seeding technique is safe, rapid, reproducible, and results in controlled quantitative cell loading, high retention, and uniform distribution.

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“…the 51 Chromium ( 51 Cr) release assay. However, this assay has been shown to have a number of disadvantages, such as high spontaneous release, inefficient labeling of target cells, a low sensitivity, and health risks associated with gamma irradiation [5,12,17,20,21,33]. Novel techniques to quantitatively and qualitatively assay Agspecific CD8 + T cells, for instance Tetramer and cytokine ELISPOT assays, have been developed during recent years.…”

Section: Introductionmentioning

confidence: 99%

OVA-specific CD8+T cells do not express granzyme B during anterior chamber associated immune deviation

Ren

Yang

et al. 2006

Graefe's Arch Clin Exp Ophthalmo

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Purpose: To examine antigen (Ag)-specific CTL response during anterior chamber associated immune deviation (ACAID). Methods: OVA or OVA257-264 peptide was injected into the anterior chamber (AC) of C57BL/6 mice. There were 16 mice in each ACAID group induced with OVA or OVA257-264 peptide. The mice were primed by SC injection with OVA or OVA 257-264 peptide in complete Freund's adjuvant (CFA) on day 7. Ag-specific CD8 + T cells in spleens were analyzed on day 14 using Pentamer H-2K b -SIINFEKL(OVA257-264 peptide). IFN-γ ELISPOT and intracellular granzyme B staining were used to characterize the CTL response. Twelve mice in each group immunized with OVA or OVA257-264 peptide in CFA served as positive controls. Twelve normal mice served as negative controls and 12 receiving injection of CFA as CFA controls for studying the influence of CFA on the Ag-specific CTL response. Result: The results showed that anterior chamber inoculation of OVA or OVA257-264 peptide could induce ACAID as evidenced by an impaired DTH response. The frequency of Ag-specific CD8 + T cells in ACAID mice was not different from that in mice challenged with Ags in CFA only (positive controls). IFN-γ production by these cells in ACAID mice was not different compared to positive controls. However, Ag-specific CD8 + T cells in ACAID mice failed to secrete granzyme B. Mice challenged only with OVA peptide and CFA also showed a granzyme B negative CD8 + T cell response. Ag-specific CTL response induced by CFA alone was similar with the negative control. Conclusion: These results show that the frequency of Ag-specific CD8 + T cells is not altered during ACAID. The Ag-specific CTL response during ACAID is characterized by the absence of granzyme B expression.

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An improved fluorescence assay for the determination of lymphocyte-mediated cytotoxicity using flow cytometry

Cited by 258 publications

References 25 publications

Implementation of the EGFP‐K562 flow cytometric NK test: Determination of NK cytotoxic activity in healthy elderly volunteers before and after feeding

Implementation of the EGFP‐K562 flow cytometric NK test: Determination of NK cytotoxic activity in healthy elderly volunteers before and after feeding

A Rapid Seeding Technique for the Assembly of Large Cell/Scaffold Composite Constructs

OVA-specific CD8+T cells do not express granzyme B during anterior chamber associated immune deviation

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