Colony-Forming Ability

These examples show that if we wanted to examine a dose-dependent decreasing function, such as survival, we might distribute some 300 colony-forming units over the 96 wells (because the commercial plates are made with 96 wells). As survival decreased from 100 to 1%, we would observe P 0) increasing from 4/96 to 93/96. With HH-4 and its clonal derivatives, plating efficiency of untreated cells in microwells is about 33%, so 300 clone-forming units are about 900 cells. 

To determine cloning ability under selective conditions, the same logic is used, but the absolute number of cells is increased, since mutants are relatively rare. To select for 6-thioguanine resistance (6TG ), where background mutant fractions are about 3 X 10 , one simply plates some 4x10 cells over 96 wells in the presence of 6TG and observes about (1/3) X (4 X 10 ) X (3 X 10 ) = 4 colonies on a 96-well plate. As the mutant fraction rises as a function of dose, the number of wells observed without colonies decreases. 

by plating 900 cells over 96 wells (one plate) in the absence of 6TG and 4x10 cells over 96 wells in the presence of 6TG, one observes the plating efficiency, PE, and the plating efficiency under selective conditions, PE tg- The mutant fraction is PE q/PE, and the effect of treatment is simply the difference between mutant fractions in the treated and untreated cultures. 

For example, suppose a culture is sampled and 900 cells are plated without selective conditions over 96 wells, and 4x10 cells are plated in the presence of 6TG. Ten wells are empty on the plate without selection, and 73 

This procedure, using microwells, is simple. Culture samples are counted and diluted to the desired concentrations (the PE dilution is best made from the PEstg dilution before adding the 6TG), selective chemicals are added to the appropriate diluted samples, and the samples are apportioned over the microwell plates. The plates are covered and placed in a 37°C, humidified, 5% CO2 incubator macroscopic colonies appear and are counted by 10-12 days. Readers who cannot handle the Poisson distributions should refer to a description of standard agar-plating protocol and are condemned to years of tedious labor,although feeder layers need not be used. 

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Studies by Hudson et al, (2000) have demonstrated the presence of eight polyphenols in rice bran by using high-pressure liquid chromatography. They are protocatechuic acid, p-coumaric acid, ferulic acid, sinapic aci vanillic acid, caffeic acid, which is a methoxycirmamic acid derivative, and tricin. The effect of these polyphenols on cell viability and on the colony-forming ability of human-derived MDA MB 468 and HBL 100 breast cells, colon-derived SW 480 and human colonic epithelial cells was assessed. These authors concluded that rice bran polyphenols have putative cancer chemopreventive properties. 

The wavelengths of light responsible for producing the recovery of colony-forming ability lie in the blue-violet and near-UV regions of the... 

Colony forming ability of the fetal liver cells was determined in the medium comprised 1.3% methylcellulose, 4.0 mM glutamine, 10 U/ml penicillin/-streptomycin, 100 U/ml GM-CSF, 100 U/ml IL-3, 50 ng/ml stem cell factor and 10 U/ml erythropoietin in IMDM. An aliquot of 10 cells was transferred to a 35 mm sterile plastic Petri dish and incubated at 37 C in a fully humidified atmosphere of 5% CO2 in air. The final colony count was performed on day 14 of culture, the colony types being defined by general morphological criteria. 

Carbonnelle et al. (1995) looked at the effect of hydroquinone on IL-1 release from human monocytes in vitro. Exposure of human monocytes to micromolar amounts of hydroquinone for 2 hours resulted in significantly decreased secretion of IL-1 a and IL-ip at doses of 5 pM and above. RNA and protein synthesis were also inhibited, with a 50% inhibitory concentration at 21 pM for IL-1 a and 10 pM for IL-1 p. Bone marrow mononuclear cells or purified hematopoietic progenitor cells were incubated with 30 pM hydroquinone alone or in the presence of IL-1 p or tumor necrosis factor, and the colony-forming ability of the cells (CFU-C) was evaluated (Colinas et al. 1995). Hydroquinone alone reduced CFU-C frequencies by approximately 60% for both bone marrow mononuclear cells and purified hematopoietic progenitor cells. Neither IL-ip or tumor necrosis factor protected the bone marrow mononuclear cells... 

Figure 3.3 The effects of sucrose pastes diluted with serum on the colony-forming ability of P. mirabilis.

Cell Survival Measurements. L1210 cell survival was quanti-fied by colony forming ability in soft agar ( ). V79 colony-... 

Denotes relative colony forming ability (no. of colonies/plating eff. X 300 no. of colonies in control/plating eff. X 300) X 100%... 

Fig. 2. Time course of PLDR in MNNG-treated cells. Quiescent cells were treated with 68 pM MNNG for 20 min and cell survival was determined by colony-forming ability at the indicated times, relative to untreated control cells. (A) Experiment 1, ( ) experiment 2...

Fig. 6. Effect of MBA on cell survival following MNNG in dividing cells. Cells were treated with MNNG MBA for 48 h and allowed to grow in control medium for an additional 48 h. The cells were reseeded for colony-forming ability. ( ) MNNG, (0) MNNG + MBA...

Incorporation of CldUrd into DNA reduces colony-forming ability to about 10% when cells are grown for 24 h in medium containing 100% CldUrd. Simultaneous treatment with SAB reduces cell survival, depending on the amount of CldUrd incorporated and concentration of SAB (Fig. 5). 

An interesting observation of the present study is that SAB effectively reduces colony-forming ability of QdUrd-substituted cells in a dose-dependent way, without affecting mutation frequency. 

Fig. 3 shows the survival of cells exposed to MNNG at various concentrations for 1 hr, followed by exposure to nalidixic acid at 100 xg for 20 hr in fresh medium. It can be observed diat nalidixic acid increased the killing by MNNG at all doses. The slope of the survival curve was also increased. Treatment of normal undamaged cells with 100 pg/ml of nalidixic acid did not affect their colony forming ability. 

FIGURE 4. Percentage survival of the colony-forming ability of strains of diploid human fibroblasts as a function of the dose of UV radiation given. See Section 2.2 for details on determining UV cytotoxicity. Taken from Maher et ai with permission. 

FIGURE 1. Effect of wild-type cell density on the observed mutant fraction and on the colony-forming ability of mutants in the presence of ouabain. All experiments were plated in microwells. (O) Butyl methanesulfonate (BMS)-treated culture (absolute mutant fraction = 2 X 10- ) ( ) clone 1 (absolute PE = 0.73) OU clone 2 (absolute PE = 0.54). 

Roberts et al. (1971) have shown that in the case of alkylating agents the degree of cellular inactivation, in terms of colony-forming ability, is best correlated to the amount of alkylation, rather than the initial dose, of the agent. Another approach is to assay the damage to transforming DNA treated in vitro as mentioned above (Mather et al., 1971). 

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