FACS analysis

Schols D, Pauwels R, Desmyter J, De Clercq E. Presence of class II histocompatibility DR proteins on the envelope of human immunodeficiency virus demonstrated by FACS analysis. Virology 1992 189 374-376. 

The fl-galactosidase complementation assay has also been adapted for use in mammalian cells (Rossi et al., 1997). The availability of fluorescent substrates for (3-galactosidase allows for fluorescence microscopy and FACS analysis of mammalian cells expressing the fusion proteins of interest. Therefore, similar to the mDHFR system, fl-galactosidase complementation assays may prove useful for genome-scale studies of protein-protein interactions in mammalian cells. 

Table 2 Cell-cycle alterations were evaluated using PI-FACS analysis and a representative result is presented in ECRF24 and A2780 cells Apoptosis rates were measured by PI-FACS analysis (n — 4) after 72 h incubation with two concentrations of drugs 1 -3...

Fig. 13.8 G1 phase arrest of HOS cells exposed to either MTX or MTX-LDH for 20 h. The cell cycle was studied by FACS analysis of Pl-stained cells. DNA histograms are shown, with the x-axis representing the DNA content and the y-axis representing the cell number. Note that 320 pg/mI MTX are equivalentto 704pg/mlof MTX-LHD, based on MTX content in MTX-LDH.

Lanier LL, Warner NL. Paraformaldehyde fixation of hematopoietic cells for quantitative flow cytometry (FACS) analysis. I. Immunol. Methods. 1981 47 25-30. 

Figure 3.10. Effect of pronase on FcyRII (CD32) and Fc yRIII (CD16) expression on neutrophils. Neutrophils were incubated in the absence (-) or presence (+) of pronase (50 pg/ml) for 30 min at 37 °C. After this incubation, expression of FcyRII and FcyRIII was determined by FACS analysis.

Figure 6.21. Role of phospholipase D in receptor up-regulation. Neutrophils were incubated in the presence or absence of fMet-Leu-Phe and butanol for 15 min prior to analysis of expression of CDllb by FACS analysis. In (a), neutrophils were not stimulated and suspensions did not contain butanol. In (b), suspensions did not contain butanol, but were stimulated with fMet-Leu-Phe. The hatched lines show the receptor expression of suspensions incubated with 10, 20 and 30 mM butanol for 5 min prior to stimulation by fMet-Leu-Phe. Source Experiment of Fiona Watson.

Figure 7.8. Role of protein biosynthesis in expression of FcyRIII. Neutrophils were incubated for (i) 15 min, (ii) 3 h and (iii) 4 h in the absence or presence of 30 /ig/ml cycloheximide. Afterwards, CD16 (FcyRIII) expression was determined by FACS analysis.

Figure 7.9. FcyRI expression in blood and synovial-fluid neutrophils. Neutrophils were isolated from the blood (trace ii) and synovial fluid (trace iii) of a patient with rheumatoid arthritis. Expression of CD64 (FcyRI) was then measured by FACS analysis. Trace (i) indicates the fluorescence distribution of a non-immune isotype control monoclonal antibody. Similar results were obtained in 6 out of 11 patients with rheumatoid arthritis.

We conducted inhibition rate analysis for each compound using human breast carcinoma cell lines (MDA-MB-231, MCF-7) and a human prostate carcinoma cell line (PC-3). Cells were treated with various concentrations of compound for 48 hr and cellular proliferation was measured by FACs analysis. 

Figure 2. Rates of apoptosis in compound treated MDA-MB-231 cells. MDA-MB-231 cells were treated with 0.1% DMSO (control) or compound (0.33 mM) for 48 hr. Annexin V and PI staining was performed followed by FACs analysis to measure apoptosis.

In Vitro Characterization and Optimization of the Liposomal Vaccine System by FACS Analysis... 

Figure 5 Relative amount of circulating TRP2 specific cytotoxic T-lymphocytes in the blood of mice. After three rounds of vaccination with the indicated formulations blood was collected and TRP2-specific CD8+ T cells were quantified by TRP2-loaded DimerX by FACS analysis. SIINFEKL-loaded DimerX used as control was a background level (data not shown).

This sol-gel procedure is an elaboration on well established entrapment methods [29], but with the added advantage of stability and better flow properties. Interestingly, none of the examples presented thus far demonstrate competitive behavior between multiple ligands (i.e. displacement) in the FAC analysis of trimethoprim and pyrimethamine a reversed order of elution based on is described, but this could simply be due to the shift towards an on-rate limited situation for higher affinity compounds, as described earlier. Erosion of dynamic competition between ligands could occur if the sol-gel allows convective mixing of the entrapped protein however the bimodal pore structure of these materials would... 

Furthermore, since the cell growth arrest is often linked to cell death. The annexin V staining positive cell or the amount of DNA fragmentation assessed by TUNEL and FACS analysis has been interpreted as indicative of apoptosis. The HDACI-induced apoptosis can also be determined by Western blotting of target proteins, detection of mitochondrial membrane potentials, activation of caspases and their substrate cleavages in a dose- and time-dependent manner. 

Newer strategies for stem cell identification have been developed based on the knowledge of cell functions. A primitive and multipotential subpopulation of bone marrow mononuclear cells has been identified on the basis of the intracellular presence of aldehyde dehydrogenase (ALDH). Those cells can be marked on the basis of the presence of ALDH and are called aldehyde dehydrogenase-bright cells (ALDH cells), allowing for their separation from a bone marrow aspiration mononuclear subpopulation under fluorescence-activated cell sorter (FACS) analysis. 

The various organs of the immune system such as spleen, lymph nodes, thymus and bone marrow containing the cells involved in the various immune responses offer the possibility to harvest these cells and perform in vitro assays for evaluation of effects on the immune system. When part of an in vivo animal study this may indicate a direct toxic effect of pharmaceuticals, that is, immunosuppression (Table 18.2). So, it is feasible to obtain cell suspensions for further evaluation such as determination of cellular subsets of T and B leukocytes by fluorescent activated cell sorter analysis (FACS analysis), and determination of natural killer (NK) cell activity of the spleen cell population. An advantage of this approach is that it may lead to identification of a biomarker to be used in clinical studies. In addition, in vitro stimulation of spleen cells with mitogens activating specific subsets may indicate potential effects on the functionality of splenic cell populations. Concanavalin A (Con A) and phytohemagglutinin (PHA) activate Tcells, while lipopolysaccharide (LPS) activates primarily B cell populations. Blood is collected for total white blood cell (WBC) determination and blood cell differential count. In addition, serum can be obtained for determination of serum immunoglobulins. 

Fluorescent Labeling of Eukaryotic Cells for Microscopy or FACS Analysis... 

Set aside 1 x 106 cell aliquots from the positive and negative cell fractions for FACS analysis. Determine total cell concentration, cell suspension volume, and population viability in the separated cell fractions. 

Expression levels of the mutant thrombin receptors were determined in transiently transfected COS cells by assaying the level of surface expressed thrombin receptors by FACS analysis, usually for mutant receptors that showed a significant diminution of function. COS cells were transfected by calcium phosphate-DNA co-precipitation30 with 10 ng of DNA. The transfected cells were analyzed by FACS 48 h after transfection. A monoclonal antibody specific for the amino terminal region was used as the primary antibody to detect thrombin receptors present on the surface of the cells.31 A FITC conjugated goat anti-mouse IgG was used as a secondary antibody. Expression levels of the mutant receptors were determined relative to expression levels of wild-type controls. Expression-level data (% of control) for some of the mutant receptors are listed in Table 2 (wild-type thrombin... 

After staining, the cells were washed once with PBS and subjected to FACS analysis. 

After 8-10 days post bone marrow transplantation, FACS analysis can be performed to monitor the development of CML in mice. Normally, at this time point, the leukemia cells (GFP+GR1+) in peripheral blood of the mice could reach to 3-5% and after 2 weeks could reach to 40-60%. 

Osella D, Mahboobi H, Colangelo D, Cavigiolio G, Vessieres A, Jaouen G (2005) FACS analysis of oxidative stress induced on tumour cells by SERMs. Inorg Chim Acta 358 1993-1998... 

F. FACS Analysis Applications in Phage Display Technology.482... 

Quantitate the fluorescence signal by FACS analysis and calculate the relative fluorescence intensity as follows (mean channel fluorescence [exp.] - mean channel fluorescence [0% control]) / (mean channel fluorescence [100% control] - mean channel fluorescence [0% control]). 

For FACS analysis of receptor expression, a gate should be set on the living cells in the forward and sideward scatter dot plots. 

Wittrup and Belting have in several studies on CPP-mediated DNA delivery established protocols for fluorescence assisted cell sorting (FACS) analysis to obtain reliable, quantitative data on CPP-DNA complex uptake in cultured cancer cells (9). Also, procedures for co-localization studies with known markers of various endocytotic pathways using confocal microscopy are described as well as the expression of dominant negative dynamin (GTPase deficient dynamin-2) to evaluate the dynamin dependence of the uptake mechanism. The use of various drugs commonly used to disrupt endocytosis is discussed, especially with regard to their limited specificity (9). 

FACS Analysis of PNA and PNA-Peptide Conjugates Cellular Uptake and Cells Permeability... 

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