LIVE/DEAD assay

Fig. 16 Fluorescence images of LIVE/DEAD assays of the L929 cells encapsulated for 4 days (a) in the miniaturized PMBV/PVA hydrogel formed in the microfluidic chip, and (b) in the bulk PMBV/PVA hydrogel formed in the 96-well microplate. Green fluorescence indicates live cells and red fluorescence indicates dead cells. Scale bar 100 pm...

Fig. 10.19 Lack of toxic effects of CM fullerene on breast epithelial cells. Cm does not inhibit cell proliferation. MCF 10A and (A) MDA MB 231 (B) breast cancer cell lines were cultured either in the absence or presence of methanol C60 and cell proliferation was assayed by crystal violet staining. Control, no C 1 Opg Cm, A 50 pg Cm, X 250 pg CM. (C). MDA MB 231 cells were simultaneously stained with calcein and ethidium using a live-dead assay kit. Lack of red-colored cells and the presence of cells stained in green indicate the lack of toxicity (D). MDA MB 231 cells were either untreated (open box) cultured with varying amounts 10 (gray ), 50 (patterned ) and lOOpg (filled ) of C60 for 48 h and analyzed for cell cycle progression by flow cytometry (Levi et al., 2006) (See Color Plates)...

Fig. 9 Viability of human endothelial cells isolated from umbilical cord on RGD modified gels (here shown for Type B) monitored by the live/dead assay. Representative pictures shows live (fluorescein di-O-acetate, viable cells visible as light spots, left) and dead (propidium iodide, no dead cells visible, right) staining. Image after 1 day culture. Scale bar 200 pm...

A similar approach was used to pattern NIH-3T3 murine embryonic fibroblasts for live/dead cell assays [855],... 

FIGURE 12.7 Representative images of human dermal fibroblasts exposed to 800pg/ml of PCL-C-DTH degradation products using LIVE/DEAD Cell Vitality assay obtained at day 42 for (a) live cells, (b) dead cells, and (c) combined image. Representative enlarged nuclei have been pointed out by arrows. Reproduced with permission from Ref. [11]. 

The standard procedure describes the growth conditions to be applied for the strain, age, and growth of the tested organism. When microbes are used the inoculum must be taken from cultures in the exponential logarithmic growth phase. This is the time when multiplication occurs and the culture is the most homogeneous. To evaluate the assay the inhibition effect has to be visualized, and the live, dead, or impeded microbes have to be distinguished. 

Live/dead cell cytotoxicity assay A measurement technique to quantify cell viability though the amount of live and dead cells within the culture. Only live cells uptake calcein which is hydrolyzed by intracellular esterases to fluoresce green. Only cells with a compromised cell membrane (dead or dying cells) can uptake ethidium which binds to DNA to fluoresce red... 

Fluorochromes can be attached to antibodies which will then bind to specific chemical structures on or inside cells. Many other chemical and physical properties of fluorochromes determine when and where these dyes are useful in various biological assays. For example, some of the fluorochromes that bind to DNA, such as Hoechst 33342, can get into living cells, but most DNA-binding fluorochromes cannot get past the cell membrane. Those fluorescent dyes that cannot get past an intact cell membrane, such as propidium iodide (PI), are often used to distinguish live from dead and dying cells. 

Niles, A., Moravec, R., and Hesselberth, E. 2007. A homogeneous assay to measure live and dead cells in the same sample by detecting different protease markers. Anal. Biochem. 366, 197-206. 

This same procedure can also be used in microscopy or FACS analysis coupled with a nuclear dye such as ethidium homodimer-1, a red fluorescent nucleic acid stain (ex/em 495/ 635 nm) that is only able to pass through the compromised membranes of dead cells, and indicates the proportion of live vs. dead cells. (The viability assay kit including calcein-AM and ethidium homodimer-1 is sold by Molecular Probes, Cat. L-3224.)... 

During the culture, the concentrations of living and dead cells (by the Trypan blue exclusion method) were measured using a haemocytometer (Chapter 2, section 2.2) glucose, lactate and glutamine by enzymic methods ammonia with a selective electrode and monoclonal antibodies by ELISA assay. 

This assay was developed and used in anti-HIV screening programs (Pauwels et al. 1988). The tetrazolium salt MTT [3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide] is converted to dark blue formazan by living cells but not by dead cells or culture medium. These assays are carried out in 96-well plates. 

The use of adenylate kinase (AK) rather than ATP for biomass estimations has been advocated/ AK is a very stable enzyme and the assay does not differentiate between living and dead cells. However, AK may be used in hygiene control. The ATP level in ADP, i.e. the AK substrate, sets the detection limit of the assay. This is a problem since ADP easily disproportionate to ATP and AMP. 

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