Incubation continuous

In one set of experiments a titration of compound is performed to assess its potency in vivo. HeLa cells are maintained in DMEM supplemented with 10% fetal bovine serum (FBS) at 37° in 5% C02. One day prior to labeling, the cells are seeded in 24-well plates at approximately 60,000 cells per well. The next day, cells are washed with warm (37°) PBS and the medium replaced with 250 41 of methionine-free DMEM containing 10% dialyzed serum (Invitrogen). After a 15-min incubation at 37°, different concentrations of compound are added to the cells (which can range from 1 nM to 50 fiM) and the incubation continued for another 45 min. Anisomycin is used as a positive control at a final concentration of 50 /iM. Fifty-five microcuries of 35S-methionine/cysteine [35S-methionine/cysteine express protein labeling mix (1175 Ci/mmol) (Per-kin-Elmer)] is added to each well (220 /(Ci/ml) and the incubation continued for another 15 min. 

Reaction with MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Sigma ) was carried out in 96-well plate at 37 °C in thermostat (Carmichael et al., 1987). The cells were incubated for 0.5, 2, and 5 h in RPMI1640 medium in the presence or absence of fullerenes C60 samples, then MTT was added, and incubation continued for 2h. The content of generated formazane was evaluated by spectrophotometry at the wavelength of X = 570 nm at the digital spectrophotometer IFCO-2 (ABOTEK, Russia). 

Although strongly suggestive, these experiments do not prove that the normal pathway of infection involves only the endocytic route. Infection could also occur by fusion of the virus with the plasma membrane. This was shown not to be the case by allowing uptake of SFV into BHK-21 cells for 10 minutes at 37°C in the presence of inhibitory concentrations of NH4CI (Helenius et al., 1982). All of the viruses left on the cell surface were then removed by proteinase K digestion at 0°C, and after removal of the inhibitor, the incubation continued at 37°C. The intracellular viruses were shown to infect the cells almost as efficiently as in control cells. 

The procedure was as follows the feedstream was made 3 M in guanidine-HCl, allowed to incubate at room temperature for 30 minutes, then dithiothreitol (DTT) was added to 5 mM and the incubation continued for one hour. Cystine was added, in 0.5 M NaOH, to 14 mM, and mixed for 10 minutes. The mixture was then diluted into 10 volumes of a 50 mM Tris buffer, pH 8.0, containing 5.3 mM cysteine. The reaction was allowed to proceed at room temperature for 16 hours (7). 

Fig. 5. Effects of preincubation of protein L with human monoclonal IgE (A.D.Z., P.S., P.P.) and human polyclonal IgG on IL-4 release from basophils. Protein L was preincubated for 15 min at 37°C with human monoclonal IgE (A.D.Z., P.S., P.P), or polyclonal IgG. Basophils were then added, and incubation continued for another 4 h at 37°C. Each bar shows the mean SEM of IL-4 release obtained from three experiments. p < 0.01 when compared with the group not preincubated with human immunoglobulin. (Reproduced with permission from Genovese et al. [80].)...

Kirkham (K2) described a very sensitive bioassay. Guinea pigs are maintained on a goitrogenic regime for 100 days the thyroids are then removed, diced, and incubated in a culture medium with TSH is added and another incubation is performed. After the second incubation an aliquot of the medium is removed and counted for radioactivity. The aliquot is replaced by KSCN and incubation continues for 4 hours, after which a second aliquot is counted. TSH increases the organification of iodine in this system, and thus the amount of nonorganic iodine discharged by KSCN into the medium is inversely proportional to the amount of TSH in the medium. 

The microorganism is cultivated in a nutrient medium without substrate for 25 h at 29r,C. 0.1 % of the substrate in alcohol is added and incubation continued at 29 °C for 103 h. The mycelial mass is acetonizcd and then extracted, together with the culture filtrate, with EtOAc. 

The test procedure is carried out over 5 days, and incubation continues for 14 more. In the first stage of the test, media-filled containers are immersed inverted into the culture of Ps. diminuta for 10 s to a depth where the container/ closure interface is completely covered. The containers are then incubated at 31°C for 24 h. Next day the procedure is repeated, but incubation of the containers over the second 24 h period is at 5 C. The third and fourth days of the trial follow the same procedure of immersion and incubation, but the containers are inverted during incubation. After immersion on the fifth day, the containers are incubated in their normal configuration at 3TC for a further 14 days. 

Three ml. of glucose oxidase solution (Worthington buffered to pH 7.0) are added, and the incubation continued for 15 minutes. One ml. of 5M HC1 is added, and the color measured at 425 m/x. The rate of production of glucose is thereby determined for each substrate. 

Bioconversion ofTHOA and DEOA. Strain ALA2 was cultivated in 50 mL of the standard medium at 30°C for 2 d, and 125 pL (100 mg) purified THOA (75% purity for 12,13, 17-THOA and 87% purity for 12,13,16-THOA) or purified DEOA (--90% pure) was added to the culture, and incubation continued. The extension of cultivation time to 2 d and the limited amount of substrate were designed to produce a better biotransformation. Intermittently, a portion (0.5 mL) of the mixture was withdrawn into a microcentrifuge tube. A 20-pL volume of 6N HCl and 0.1 mL of 10 mg/mL palmitic acid in ethylacetate (internal standard) were added. The lipids were extracted twice with 0.5 mL of ethylacetate, dried under nitrogen gas, and converted into methyl esters using diazomethane. 

Incubation continued with routine sampling until all added substrate has been biotransformed to products(s)... 

Fig. la,b. Nucleoid sedimentation analysis. HeLa cells were grown for 24 h at 37°C in the presence of [6- H] thymidine at 1 fiCi mf (sp.act. 26 Ci mmol" ). The medium was then removed and replaced with fresh medium (a) or with medium containing 100 mA/ methotrexate (b) and incubation continued for 1 h. Subsequent nucleoid sedimentation analysis (6000 rpm for 30 min at 20°C, SW50.1 rotor) was as described by Cook and Brazell [3]. Fractions were collected from the bottom of gradients and precipitated onto GF/C filters for radioactivity determination. Sedimentation is from right to left... 

At 4 hf 2 mCi of (55s) methionine were added, and the incubation continued to 6.5 h. The cells were collected and resuspended in a medixmi containing cold methionine. After a 1 h chase the cells were harvested and from them we prepared a whole cell lysate, isolated the smooth cytoplasmic membranes and from these purified the replicase by QAE Sephadex chromatography and glycerol gradient sedimentation. Samples of radioactive cell lysate, smooth membrane and purified enzyme were analyzed by SBS polyacrylamide gel electrophoresis. A profile of the radioactive proteins in a cell lysate is shown in Figure 10A. It contains three minor peaks of molecular weight 95>000> 75>000 and 65,000 which correspond to EMC virus unstable proteins B,B and Bl three of the four coat proteins a (34,000), 3 (30,000) and y (26,000) and relatively large amounts of stable EMC virus proteins E (56,000) and F (38,000). A profile of the EMC proteins found in the isolated smooth membranes is shown... 

Various concentrations of nonradioactive nucleotide analogues were incubated with 50 pi of antiserum (final dilution of 1 150) overnight at 0-4°C in volume of 0.4 ml PBS-BT. pH]NAD tracer was added (- 30,000 cpm) in 50 pi and the incubation continued for another 24 hrs. Antibody bound tracer was separated from unbound fraction by DCC treatment Controls were set up with no antiserum and with no competing ligands. 

The lymphocyte fraction is separated from heparinized blood by Ficoll gradient centrifugation and the lymphocytes adjusted to a cell density of 10 cells/ml. For prestimulation of the lymphocytes, phytohemagglutinin (PHA-M) or concanavalin A are used as mitogens. The prestimulated lymphocytes together with the substance to be tested are transferred to microtitre plates and incubated for 70 or 80 h. H-thymidine is added and the incubation continued for a further 18 h. The quantity of H-thymidine incorporated is determined by scintillation counting (cpm). Percent stimulation is taken as the difference between the incorporated H-thymi-dine of the test and control incubations, multiplied by 100, and divided by the control value. 

Fic. 7. The effect of P5C on PP-ribose-P as a function of phosphate concentration. Erythrocytes were preincubated for 30 minutes in 145 mMNaCl, 10 mMTris-HCl, pH 7.4,1.2 mMMgCh, and varying concentrations of sodium phosphate, pH 7.4. After preincubation 2.5 mM glucose and 0.5 P5C ( ) or glucose alone (O) were added and the incubation continued for 1 hour. Adapted from ref. (118). 

Leukocytes from blood are suspended (4% by volume) in 0.1 ml of Fischer s medium containing 25 mM phosphate buffer, pH 7.4, and incubated for 20 minutes. Radioactive adenine, guanine or hypoxanthine (50 yM final concentration) are added to separate tubes and incubation continued for 3 hours. For suspension cultures,. 

Fig. I6a-c. Inhibition of host cell protein synthesis by addition of DEAE-dextran, DM SO or salt. HeLa cells from a suspension culture were harvested by centrifugation, resuspended in Eagle s medium without serum at 4 X10 cells/ml and incubated at 37° C for 10 minutes, then 10 (xCi/ml of leucine (a, b) or 2.0 (xCi/ml of amino acids were added and incubation continued for 5 minutes. Portions of the cell culture were transferred to small tubes containing saline (control), different amounts of DM SO...

Fig. 17. Effect of DEAE-dextran on the growth of E, coli. E. coli MRE 600 was grown in Vogel-Bonner medium to a density of 3-5 X 10 cells/ml. DEAE-dextran was added to final concentrations as indicated below and incubation continued at 37° C. At 0 time...

Fig. 2. Effect of chloramphenicol, chlortetracycline and puromycin on incorporation of glycine-l- C into protein and edeine when added in late logarithmic phase. After 8 hr of incubation under the conditions described in Fig. 1, cultures were supplemented with 100 (xg/ml of chloramphenicol, 10 xg/ml of chlortetracycline or 100 xg/ml of puromycin and incubation continued for 45 minutes, (x) control ( ) with chloramphenicol (o) with chlortetracycline (A) with puromycin...

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