Flow cytometric

Figure 5B. Correlation of right-angle light scatter measured by fluorometry and flow cytometry. The top panel shows flow-cytometric data of side scatter of fixed, stained cells during the time course of stimulation by 1-nM (solid line, solid circles) or 0.01-nH (dashed line, open circle) FLPEP. The bottom panel shows the corresponding right-angle light-scatter data acquired pseudo-simultaneously on live cells in the fluorometer. The flow-cytometric data have been averaged, but the fluorometry data are plotted for both duplicates from one donor. Reproduced with permission from Ref. 27. Copyright 1985 Rockefeller University Press.

Hence, pseudosimultaneous spectroscopic and flow-cytometric measurements suggest that actin polymerization may be functionally related to aggregation, possibly by reorganization of adhesive molecules on the cell surface. Simultaneous spectroscopic and fluorometric measurements suggest that the signal Ca may be related to oxidant production and degranulation. 

Calibration. Many approaches have been used to calibrate flow cytometric measurements. Including the comparison of flow and nonflow techniques (radiolabels, spectrofluorometry). In recent years, commercial standards have been introduced which are calibrated in fluorescein equivalents/particle (e.g., 3,000 or 500,000). With labeled ligands, calibration requires determining the relative quantum yield of the ligand compared to pure fluorescein and using the standards to analyze the amount bound on cells. Our ligands (fluorescein isothiocyanate derivatives) are typically 50% as fluorescent as fluorescein. 

Sanz ML, Gamboa PM, Antepara I, Uasuf C, Vila L, Garda-Aviles C, Chazot M, De Week L Flow cytometric basophil activation test by detection of CD63 expression in patients with immediate-type reactions to (J-lactam antibiotics. Clin Exp Allergy 2002 32 277-286. 

Gamboa P. Sanz ML. Caballero MR. Urrutia I. 43 Antepara I. Esparza R. De Week L The flow-cytometric determination of basophil activation induced by aspirin and other non-steroidal and anti-inflam- 44 matory drugs (NSAIDs) is useful for in vitro diagnosis of the NSAID hypersensitivity syndrome. Clin dS Exp Allergy 2004 34 1448-1457. 

Sudheer PS, Hall JE, Read GF, Rowbottom AW, Williams PE Flow cytometric investigation of peri-anaesthetic anaphylaxis using CD63 and CD203c. Anaesthesia 2005 60 251. 

Flow cytometric evaluation of bone marrow and peripheral blood to characterize the type of leukemia, as well as to detect specific chromosomal rearrangements. The bone marrow at diagnosis usually is hypercellular, with normal hematopoiesis being replaced by leukemic blasts. The presence of greater than 20% blasts in the bone marrow is diagnostic for AML. 

Borzi RM, Mazzetti I, Macor S, et al. Flow cytometric analysis of intracellular chemokines in chondrocytes in vivo constitutive expression and enhancement in osteoarthritis and rheumatoid arthritis. FEBS Lett 1999 455(3) 238-242. 

Kuhne T., Homstein A., Semple J., et al. Flow cytometric evaluation of platelet activation in blood collected into EDTA vs. Diatube-H, a sodium citrate solution supplemented with theophylline, adenosine and dipyridamole. Am J Hematol 1995 50,40-5. 

Apoptosis assay. ECRF24 or A2780 cells were seeded on 6-well plates (2 X 105 cells/ well) and grown 24 hours in complete medium before treatment. Compounds 1-3 were freshly dissolved in DMSO, diluted in complete medium and added to the cells at the final concentrations indicated in Table 2. After incubation for 72 h apoptosis was measured by flow cytometric determination of subdiploid cells after DNA extraction and subsequent staining with propidium iodide (PI) as described previously10. Briefly, cells were harvested and subsequently fixed in 70% ethanol at —20°C. After 2 h the cells were re-suspended in DNA extraction buffer (45 mM Na2HP04, 2.5 mM citric acid, and 1% Triton X-100, pH 7.4) for 20 min at 37°C. PI was added to a final concentration of 20 pg/mL and log scale red fluorescence was analyzed on a FACSCalibur (BD Biosciences, NJ, U.S.). 

Fuchs, B. M. Wallner, G. Beisker, W. Schwippl, I. Ludwig, W. Amann, R. Flow cytometric analysis of the in situ accessibility of Escherichia coli 16S rRNA for fluorescently labeled oligonucleotide probes. Appl. Environ. Microbiol. 1998, 64, 4973 1982. 

Braga, E C. Bovio, C. Culici, M. Dal Sasso, M. Flow cytometric assessment of susceptibilities of Streptococcus pyogens to erythromycin and rokitamycin. Antimicrob. Agents Chemother. 2003, 47, 408-412. 

Holm, C. Jespersen, L. A flow-cytometric gram-staining technique for milk-associated bacteria. Appl. Environ. Microbiol. 2003, 69, 2857-2863. 

Tron, L., Szollosi, J., Damjanovich, S., Helliwell, S. H., Arndt-Jovin, D. J. and Jovin, T. M. (1984). Flow cytometric measurement of fluorescence resonance energy transfer on cell surfaces. Quantitative... 

Szabo, A., Horvath, G., Szollosi, J. and Nagy, P. (2008). Quantitative characterization of the large-scale association of ErbBl and ErbB2 by flow cytometric homo-FRET measurements. Biophys. J. 95, 2086-96. 

For cell cycle analysis, HOS cells (1 x 106) were treated with the indicated concentrations (see Table 13.2) of MTX or MTX-LDH for 20h, harvested by trypsin treatment, washed with PBS, and then fixed with cold 70 % ethanol on ice overnight. The fixed cells were incubated with propidium iodide and flow cytometric measurement was carried out. Over 20 h, an increase in the number of cells in the Gl phase resulted from MTX and MTX-LDH treatment compared to untreated cells, indicating arrest at the Gl/S boundary. It is worth noting that the inhibition of the Gl/S transition was more evident in the cells treated with MTX-LDH than in those treated with free MTX (85.59% versus 66.62% at 320pM/ml). This is consistent with... 

Reader, S., H.B. Steen, and F. Denizeau. 1994. Intracellular calcium and pH alterations induced by tri-n-butyltin chloride in isolated rainbow trout hepatocytes a flow cytometric analysis. Arch. Biochem. Biophys. 312 407-113. 

Gala, W.R. and J.P. Giesy. 1994. Flow cytometric determination of the photoinduced toxicity of anthracene to the green alga Selenastrum capricomutum. Environ. Toxicol. Chem. 13 831-840. 

George, L.S., C.E. Dallas, I.L. Brisbin, Jr., and D.L. Evans. 1991. Flow cytometric DNA analysis of ducks accumulating 137Cs on a reactor reservoir. Ecotoxicol. Environ. Safety 21 337-347. 

Compounds that are not overtly myelotoxic may still selectively damage or destroy lymphocytes, which are the primary effectors and regulators of acquired immunity. This toxicity may result from the destruction of rapidly dividing cells by necrosis or apoptosis. A variety of methodologies are available for this purpose (e.g., colorimetric, flow cytometric assays). If the cells are viable (80% or greater), basic functionality could be determined by performing specific functional assays. 

Filippini, G. et al., Flow cytometric detection of p53 protein after incubation of a pre-B cell line with antitumor agents, Cytometry, 35, 267, 1999. 

June, C.H. and Rabinovitch, P.S., Flow cytometric measurement of intracellular ionized calcium in single cells with indo-1 and fluo-3, Methods Cell Biol., 33, 37, 1990. 

Fischer, K. and Mackensen, A., The flow cytometric PKH-26 assay for the determination of T-cell mediated cytotoxic activity, Methods, 31, 135, 2003. 

Pestka, J. J., Yan D., and King L.E. Flow cytometric analysis of the effects of in vitro exposure to vomitoxin (deoxynivalenol) on apoptosis in murine T, B and IgA+ cells. Food Chem. Toxicol. 32, 1125, 1994. 

Keij J, Steinkamp J (1998) Flow cytometric characterization and classification of multiple dual-color fluorescent microspheres using fluorescence lifetime. Cytometry 33 318-323... 

Koyama, S., Maruyama, T., and Adachi, S. (1999). Expression of epidermal growth factor receptor and CD44 splicing variant sharing exons 6 and 9 on gastric and esophageal carcinomas a two flow-cytometric analysis. J. Cancer Res. Clin. Oncol. 125, 47—54. 

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