A follow‐up study of the development of Rauscher erythroleukemia,

Szabó, Gábor; Mátyus, László

doi:10.1002/cyto.990050114

Cited by 3 publications

(1 citation statement)

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“…The relatively small spectral overlap allows one to detect these dyes independently. (108)(109)(110) Tetramethylrhodamine isothiocyanate (TRITC) can be excited very well with mercury arc lamps (565-nm line), and while the 514-nm line of an argon ion laser is not optimal, it gives satisfactory results. For multiparameter studies, when more than one surface antigen is labeled at a time, other dyes are necessary.…”

Section: Immunofluorescence Measurementsmentioning

confidence: 99%

Flow Cytometry and Cell Sorting

Mátyus

Edidin

Topics in Fluorescence Spectroscopy

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Section: Immunofluorescence Measurementsmentioning

confidence: 99%

Flow Cytometry and Cell Sorting

Mátyus

Edidin

Topics in Fluorescence Spectroscopy

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Fluorescent staphylococci as microbeads

Szabó

Damjanovich

1989

Cytometry

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Fixed bacteria of the protein A-rich Co-results are documented with fluoreswan I Staphylococcus strain were la-cence microscopy and flow cytometry. belled with fluorescein isothiocyanate and used as the second-step reagent in an indirect immunofluorescent assay of specific cell-surface antigen expression. The Key terms: Cytometry, microbeads, protein A Application of antibody-carrying fluorescent microbeads is a very attractive approach in immunofluorescent cell-surface labelling experiments. An increased sensitivity can generally be expected due to a high fluorochromelantibody ratio (for successful application, see ref. 9). A limitation to the widespread application of microbead technology comes from the difficulties in coupling proteins to the microspheres (1, 4) and from the need for large quantities of purified antibody (or protein A). These difficulties seem to be shared by the method that uses carboxylfluorescein-filled, protein A-coated liposomes to detect low receptor numbers (8).We attempted to use fluoresceinated Staphylococcus bacteria, strain Cowan I, wearing a cell-coat abundant in protein A (for review, see ref. 21, as second-step microbeads. MATERIALS AND METHODSFixed bacteria ("Pansorbin cells") are available from Calbiochem Behring Co.; 1 ml of the 10% cell suspension was washed three times with phosphate-buffered saline (PBS), suspended in a fluorescein isothiocyarate (FITC) solution (1 mg/ml, dissolved in PBS, pH 7.4) and incubated for 1.5 h a t 37°C. The bacteria were washed three times with PBS and dialysed against PBS + 0.02% sodium azide for 24 h. Cell concentration was set to approximately 5%.To test the product, Rauscher virus-infected Balb/c mouse spleen cells were labelled (6) with virus envelope glycoprotein (gp70)-specific hyperimmune goat serum (anti-gp70 [see ref. 51, a kind gift of Dr. H. Schwarz, Max-Planck Institut fur Virusforschung, Tubingen), washed twice in a refrigerated centrifuge with ice-cold PBS (5). The pellet of about 1 x lo6 antigp70-labelled cells was suspended in 50 p1 of cold PBS, mixed with the same volume of labelled bacteria, and kept on ice for 15 min with frequent shaking of the test tube. After washing twice with a n excess of cold PBS, the final cell pellet was suspended in 1 ml of PBS and analysed with a Becton-Dickinson FACS I11 flow cytometer. The argon-ion laser was tuned to 488 nm, and fluorescence was detected through a 510-nm cutoff filter. Figure 1A shows that spleen cells 9 days after inoculation of the virus express anti-gp70-reactive antigens in amounts easily detectable with this method. The approximately 30-fold difference between the samples incubated with and without serum exceeds the approximately 15-fold difference obtained with a standard indirect immunofluorescent assay on the same cells. In terms of coefficient of variation, however, our method gives wider distributions than the standard assay. We did not observe any toxic effect of the bacteria on cells incubated either with or without serum (data not shown). Figure 1B shows the fluores...

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