Cell staining

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.

Fig. 15. Effects of turbulent shear stress level and exposure time on cell viability measured by trypan blue staining. Cells were sheared in a concentric cylinder viscometer [1]...

Cells Incubated with Percentage of Stained Cells Protection Factor... 

Calcein AM (CAM) Live cells green stained Cell membrane Yesa [34]... 

Trypan Blue exclusion (TB) Dead cells blue stained Cell membrane Yes3 [34]... 

Direct division of the chromatin bodies of E. coli has been demonstrated by Mason and Powelson in a series of remarkable phasecontrast photomicrographs. These observations were made on living bacteria. The nuclear areas in the dividing cells appeared to be as clearly defined as the areas in fixed, hydrolyzed, and stained cells. 

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.

Figure 10.2 An immunostained specimen, after segmentation for the stained cell nuclei. In this specimen, one can either count the nuclei or divide the total segmented white area by an average nuclear area (taking into account few nuclei are full size). 

Light and electron microscopic studies were performed on the synovial membranes (E2) of patients with HIV associated arthropathy. An immunoperoxidase technique with the use of monoclonal antibodies against CD4, CD8, B, and DR lymphocytes and HIV p 24 antigen was also used. Mild to moderate nonspecific proliferative changes and increased vascularity of the subsynovial space were seen. Immunohistochemical staining revealed HIV p 24 positive staining cells of the synovial lining layer and the mononuclear cells of the subsynovial space, CD4, CD8 with predominance of CD8, B, and DR cells were also present. 

Fig. 11.2 (a) HAADF-STEM image of a stained cell section (40nm thick). A SWNT cluster within a lysosome invading the lysosomal cell membrane, (b) Corresponding high-resolution lattice image of SWNTs at the lysosomal membrane from boxed area. Cytoplasm (cy) and secondary... 

The problem has now been solved, and it is possible to measure the phase angle of the probe as the cells pass through the laser beam.09,40) While these measurements have not yet been applied to Ca2+, the method has been validated with standard beads and stained cells. In our opinion, this new flow cytometry parameter, and our increasing knowledge of lifetimes of probes, will result in the increased use of flow cytometry for studies of intracellular physiology, in addition to the current emphasis in immunology, cell activation, and ploidy. 

Before further testing and to confirm that the compounds are cytotoxic rather than merely interfering with the Alamar blue indicator dye, they are re-bioassayed using two other indicator dyes. Calcein-AM is a fluorescent dye that measures changes in cell mem brane permeability, an indicator for one of the penultimate steps of cell death, uci e e measures the amount of adenosine triphosphate (ATP) synthesis in a chemilurmne assay. For some compounds, cell death was also confirmed by microscopic exami Papanicolaou-stained cell preparations.11... 

Load 20 p.1 of the stained cells through the sample loading area by capillary action (Fig. D.4). 

It is useful to resuspend the stained cell pellet in 1 mL PBSG with 500 ng/mL propidium iodide (PI) to detect dead cells by the inclusion of PI and its resultant red fluorescence on the flow cytometer. Red fluorescence (PI) vs light scatter is used to exclude dead cells before collecting green (FITC) emitting cells of the viable cell population. 

Staining Cell-Surface Antigens Prior to Fixation for Flow Cytometric Analysis... 

Fig. 3. Micrograph demonstrating die effect of NaSal on morphological changes in eosinophils (W16). After eosinophils were treated for 12 h (a) without or (b) with 20 mM NaSal, which is an apoptosis-inducing agent, cells were harvested and stained with Hemacolor Rapid blood smear staining set. The stained cells were examined by light microscopy. The arrows depict the apoptotic eosinophils...

Analyze a sample of the most strongly stained cells in the experiment. 

On the FL2 vs FL3 plot, adjust the FL3 - FL2 compensation to move the PE-Cy5-stained cells from spilling into the FL2 scale (Fig. 2G), to run parallel to... 

On the FLI vs FL3 plot, note that there should be no spillover of FL3 into FLI (Fig. 21), similarly, there should be no spillover ofFLl-stained cells into the FL3 scale (not shown)... 

Another important limitation to be aware of is that the induction of apoptosis in a population of cells will not always result in a clearly distinct subdiploid peak (12), We have seen this particularly when apoptosis is induced in Burkitt lymphoma cells by serum deprivation. After approx 2 wk, all cells are apoptotic as can be clearly seen by the two-dimensional light scatter assay and confirmed by microscopic analysis of acridine orange-stained cells, but the cell-cycle profile gives the appearance of a mainly viable population (Fig. 5). 

---