DMEM

FCS, fetal calf semm and DMEM, Dulbecco s Eagles minimum... 

Eagle s MEM with serum rapidly became a standard growth medium for culturing animal cells in vitro. A number of variations of this medium were developed, including Dulbecco and Vogt s modified Eagle s essential medium (DMEM) (Table 2). DMEM contains nonessential as well as essential amino acids. The essential amino acids and vitamins are at concentrations which are significantly elevated as compared to MEM. 

Tumor cells. EMT6 cells were grown as a monolayer culture in DMEM medium containing 20% fetal calf serum (27). Cells were detached from the plate by trypsin-EDTA treatment and washed in PBS. A total of 5 x 103 cells were injected per mouse via the tail vein of Balb/c mice (6-8 weeks old) to induce experimental lung metastatic tumors. Immunoliposomes were injected iv 2 and 4 days after the tumor cell injection. The survival of mice was followed over the next 60 days. 

However, when the I AM log were compared to liposome log Dmem, 7.0,... 

GETTING IT WRONG FROM ONE-POINT log Dmem MEASUREMENT... 

Place a coverslip in a 6-well plate and seed cells in 2 ml DMEM medium (plus 10% FCS, glutamate, penicillin, and streptomycin). 

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. 

One day prior to the isolation of polysomes, approximately 5 million HeLa cells are plated into 10-cm dishes. The following day, the media is replaced with fresh DMEM and compound is added to a concentration previously determined to inhibit translation in vivo by 35S-methionine metabolic labeling (see previously). 

The next day (48 h posttransfection), the medium is replaced with fresh DMEM containing different concentrations of compound (nM—fiM concentration range). After 12 h of incubation, the cells are washed with PBS, 40 1 of luciferase lysis buffer [100 mM KxP04 (pH 7.8), 0.2% Triton X-100] is added to each well, and the plate is incubated for 15 min at room temperature with gentle rocking. The cell extract is transferred into Eppendorf tubes and kept on ice. 

RKO cells were cultured in DMEM medium (high glucose) supplemented with fetal bovine serum and antibiotics (Invitrogen) in eight 150 x 25 mm cell culture dishes to 50 to 60% confluency. Upon removal of... 

Fig. 2.2 Stability of IgCi monoclonal antibody added to sterile plant and animal cell culture media. ( ) Murashige and Skoog (MS) medium (A) Dulbecco s minimal essential medium (DMEM) with 10% serum and (A) serum-free Ex-cell 302 medium. The error bars indicate standard errors from triplicate flasks. (Reproduced with permission, from B. M. -Y. Tsoi and P. M. Doran, Biotechnol. Appi. Bio-chem. 2002, 35, 171-180. Portland Press on behalf of the IUBMB.)...

Fig. 17. Confocal fluorescence imaging of [Zn(ATSM)] in IGROV cells (100 pM, where Q1 — Q2 = Me, M = Zn(II), R1 = R3 = H and R2 = R4 = Me, /ex = 488 nm, DMEM with 1% DMSO). Brightfield image shows formation of needle-like crystalline material on the cell plate (N.B. Small crystallites may be endocytosed by the cells rather than passively diffuse through the cell membrane).

Fig. 21. Molecular structures of new aromatic [M(ATSM)] analogs (a) M — Zn(II) and (b) M = Cu(II), (c) cytotoxicity tests in MCF-7 cells for the Zn(II) complex (group 2) and Cu(II) complex (group 3) and comparison with control and with cis-platin over a range of concentrations, (d) cell uptake profile monitored over 90 min, (e) confocal fluorescence imaging of Zn(II) complex in MCF-7 cells, at 100 pM cone, in DMEM, 1% DMSO (112,113).

Cell lines U937 (human lymphoblastoid cells) and T-98G (human astrocytoma) were obtained from the collection of cell lines at the Influenza Research Institute. T-98G cells were grown on a DMEM medium with the addition of 10% calf serum, and U937 cells onRPMI1640 (Biolot, St. Petersburg) with 10% calf serum. 

Figure 4 Effect on the serum stability of the incorporation of 5% cholesterol poly(ethy-lene glycol) (PEG) into cationic lipoplexes [hpopolyamine RPR209120/DOPE1/1, ratio (mol) lipid/DNA = 10 in 150 mM NaCl]. Lipoplexes were incubated in DMEM + 10% SVF, at 37°C, aliquots were regularly sampled and monitored by dynamic diffusion. Results represent a mean between three measurements. Error bars are not presented to simplify the graph, but differences among PEG, PEG-1, and PEG-2 are significant. Abbreviations PEG, poly(ethylene glycol) DOPE, dioleylphosphatidylethanolamine DMEM, Dulbecco s Modified Eagle Medium.

For incubation, cells are preincubated for five minutes at 4°C in culture medium without buffer substances and acidified with 20 mM succinic acid, pH 5.7 [Dulbecco s modified Eagle s medium (DMEM) without sodium bicarbonate, but containing 0.6% bovine serum albumin (BSA), 20 mM 2-[N-morpholino]-ethanesulfonic acid (MES), 20 mM succinic acid, pH 5.7)]. For different methods useful for acidification of cells, see Ref. (43). 

Shown in Figure 6 is in vitro cytotoxic activity of PIPAAm-PBMA micelles loaded with ADR or micelles without ADR at 29°C (below the LCST) and at 37°C (above the LCST) compared with that of free ADR. In vitro cytotoxic activity was measured using bovine aorta endothelial cells. Bovine aortic endothelial cells were obtained as previously reported using dispase for cell dissociation from freshly harvested bovine aorta [13]. The cells plated at a density of 3 x cells/well, were exposed with free ADR or micelles loaded with ADR at below and above the LCST for 5 days. In order to assay cytotoxicity of the free ADR or micelles loaded with ADR, culture medium was replaced with 10% FBS-supplemented phenol red-free DMEM containing 10% alamar Blue, a dye that is subject to reduction by cytochrome c activity and changes the color from blue to red [38]. After 4-hour incubation, reduction of the dye was estimated by absorbance at 560 and 600 nm. PIPAAm-PBMA polymeric micelles loaded with ADR showed higher cytotoxic activity than that of free ADR at 37°C (above the LCST)... 

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