Carboxyfluorescein fluorochromasia assays. I. Non-radio actively labeled cell mediated lympholysis

Bruning, J. W.; Kardol, M. J.; Arentzen, René

doi:10.1016/0022-1759(80)90080-0

Cited by 110 publications

(48 citation statements)

References 8 publications

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“…The use of fluorescent labels to measure cell-mediated cytotoxicity has been employed for a number of years (9,31). A fluorochrome assay using the carboxyfluorescein derivative 2 ¶,7 ¶-bis(carboxymethyl)-5,6-carboxyfluorescein was reported to be similar to the frequently used 51 Cr release assay (31).…”

Section: Discussionmentioning

confidence: 99%

“…A fluorochrome assay using the carboxyfluorescein derivative 2 ¶,7 ¶-bis(carboxymethyl)-5,6-carboxyfluorescein was reported to be similar to the frequently used 51 Cr release assay (31). Another fluorescent dye, fluorescein diacetate, is converted in viable cells by intracellular esterases to fluorescein, which becomes trapped within cells having intact membrane integrity (15), causing the viable cells to exhibit bright green fluorescence.…”

Section: Discussionmentioning

confidence: 99%

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A fluorescence microplate cytotoxicity assay with a 4-log dynamic range that identifies synergistic drug combinations

Frgala

Kalous

Proffitt

et al. 2007

Molecular Cancer Therapeutics

107

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Purpose: Cytotoxicity assays in 96-well tissue culture plates allow rapid sample handling for multicondition experiments but have a limited dynamic range. Using DIMSCAN, a fluorescence digital image system for quantifying relative cell numbers in tissue culture plates, we have developed a 96-well cytotoxicity assay with a >4-log dynamic range. Methods: To overcome background fluorescence that limits detection of viable cells with fluorescein diacetate, we used 2 ¶4 ¶5 ¶6 ¶-tetrabromofluorescein (eosin Y) to quench background fluorescence in the medium and in nonviable cells to enhance the reduction of background fluorescence achieved with digital image thresholding. The sensitivity and linearity of the new assay were tested with serial dilutions of neuroblastoma and leukemia cell lines. DIMSCAN was compared with other in vitro cytotoxicity assays: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, colony formation, and trypan blue dye exclusion. Results: Without background fluorescence reduction, scans produced a nearly flat curve across various cell concentrations from 100 to 10 6 cells per well. Either digital image thresholding or eosin Y dramatically reduced background fluorescence, and combining them achieved a linear correlation (r > 0.9) of relative fluorescence to viable cell number over >4 logs of dynamic range, even in the presence of 4 Â 10 4 nonviable cells per well. Cytotoxicity of deferoxamine for neuroblastoma cell lines measured by the DIMSCAN assay achieved dose-response curves similar to data obtained by manual trypan blue counts or colony formation in soft agar but with a wider dynamic range. Long-term cultures documented the clonogenic ability of viable cells detected by DIMSCAN over the entire dynamic range. The cytotoxicity of two drug combinations (buthionine sulfoximine + melphalan or fenretinide + safingol) was tested using both DIMSCAN and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, and the wider dynamic range of DIMSCAN facilitated detection of synergistic interactions. Conclusion: DIMSCAN offers the ability to rapidly and efficiently conduct cytotoxicity assays in 96-well plates with a dynamic range of >4 logs. This assay enables rapid testing of anticancer drug combinations in microplates. [Mol Cancer Ther 2007; 6(3):886 -97]

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

A fluorescence microplate cytotoxicity assay with a 4-log dynamic range that identifies synergistic drug combinations

Frgala

Kalous

Proffitt

et al. 2007

Molecular Cancer Therapeutics

107

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“…The software and assay systems have been originally pionieered by Bruning [1]. We compared its reliability with the conventional serological microcytotoxicity assays for HLA class I and class II typing [2].…”

Section: A Wimmer a Graf R Wankmentioning

confidence: 99%

Abstracts (Part 1 of 6)

1991

Transfus Med Hemother

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“…In the granulocytotoxicity assay [8], unfixed granulocytes were stained with green fluorescent carboxyfluorescein [9]. After incubation with the test serum for 60 min at 20 °C and with properly diluted complement for 90 min at 20 °C, the dead cells were counterstained with red fluorescent ethidium bromide (Sigma).…”

Section: Granulocytotoxicity Testmentioning

confidence: 99%

Group-Specific Auto-Immune Antibodies Directed to Granulocytes as a Cause of Chronic Benign Neutropenia in Infants

Sabbe¹,

Claas²,

Langerak³

et al. 1982

Acta Haematol

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Chronic benign neutropenia of infancy is a disease which develops a few months after birth and which is characterized by a severe selective neutropenia, accompanied by benign but persisting infections. The cause of the disease is still unknown. The sera from 5 such patients were tested for the presence of granulocyte antibodies as a possible cause of the disease. For the detection of these antibodies immunofluorescence, agglutination and granulocytotoxicity were used. All sera contained antibodies which reacted both with the neutrophils of one or both parents of the patient and a part of a panel of unrelated donors. From the reaction patterns against the panel we could identify the specificity of three sera. Two sera were directed to the neutrophil-specific antigen NA2, and the third one reacted with a hitherto not yet recognized neutrophil-specific alloantigen which we called NE1. In 4 patients we could confirm the autoimmune character of the disease by demonstrating the antibody on the patients’ own granulocytes. These results suggest that autoimmunity may be the cause of many cases of benign infantile neutropenia.

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Carboxyfluorescein fluorochromasia assays. I. Non-radio actively labeled cell mediated lympholysis

Cited by 110 publications

References 8 publications

A fluorescence microplate cytotoxicity assay with a 4-log dynamic range that identifies synergistic drug combinations

A fluorescence microplate cytotoxicity assay with a 4-log dynamic range that identifies synergistic drug combinations

Abstracts (Part 1 of 6)

Group-Specific Auto-Immune Antibodies Directed to Granulocytes as a Cause of Chronic Benign Neutropenia in Infants

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