Cell culture medium preparation

Autofluorescence of cells often complicates the studies with fluorescence microscopy (especially the application of green fluorescent substances). There are different reasons for the occurrence of this phenomenon (157) (i) the fluorescent pigment lipofuscin, which settles with rising age in the cytoplasm of cells (ii) cell culture medium, which often contains phenol red that increases autofluorescence (iii) endogen substances such as flavin coenzymes [flavin-adenine dinucleotide (FDA), flavin mononucleotide (FMN) absorp-tion/emission 450/515nm], pyridine nucleotides [reduced nicotinamide adenine dinucleotide (NADH) absorption/emission 340/460nm] or porphyrine (iv) substances taken up by cells (as mentioned above filipin) and (v) preparation of the cells fixation with glutaraldehyde increases autofluorescence. 

Hepes is used at 10-25 mM and is added to medium from a stock solution (1 M) whose preparation is described in Appendix 1, Table A1.5. As well as Hepes, other zwitterionic buffers have been used in cell culture medium. TRICINE (lV-[Tris-hydroxymethyl)-methyl]gly-cine, pKa = 7.79 at 37°C) has been used in Eagle s MEM (Spendlove et al., 1971) and in Swim s 577 (Gardner, 1969) and TES, (iV-[(Tris-hydroxymethyl)methyl]-2-aminoethanesulphonic acid, pKa = 7.16 at 37°C) has been used in Eagle s MEM, BME and in medium 199 (Williamson and Cox, 1968 Massie et al., 1972). These buffers are used in varying concentrations (10-50 mM). Eagle (1971) suggests combinations of buffers which can be used to buffer the medium over the range 6.4-8.35. 

An IR spectrum of freshly prepared RPMI 1630 cell culture medium supplemented with 5% calf serum, the medium used routinely to culture viable cells, is presented in Figure 1. Figure 1 is an IR spectrum of centrifugally concentrated and washed EAT cells which were cultured in this medium. The IR spectrum of the salt-free residue from the supernatant liquid obtained after centrifuging the cells constitutes Figure... 

Figure 1. IR spectrum of freshly prepared RPMI 1630 cell culture medium (+5% calf serum supplement) with no cells present...

Since a mammalian cell culture medium was first prepared by Earle et al. many different kinds ofbasal media have been established. For example. 

Normal animal tissues (e.g., skin, kidney, liver) or whole embryos are commonly used to establish primary cell cultures. To prepare tissue cells for a primary culture, the cell-cell and cell-matrix interactions must be broken. To do so, tissue fragments are treated with a combination of a protease (e.g., trypsin or the collagen-hydrolyzing enzyme collagenase or both) and a divalent cation chelator (e.g., EDTA) that depletes the medium of usable Ca or Mg. The released cells are then placed in dishes in a nutrient-rich, serum-supplemented medium, where they can adhere to the surface and one another. The same protease/chelator solution is used to remove adherent cells from a culture dish for biochemical studies or subculturing (transfer to another dish). 

In preparing fixatives, measure the concentration of particles (non-water molecules) in a solution. The units of measure here are osmoles. A reading of 300 mOsm is the normal physiological concentration inside cells, in cell culture medium, and in blood serum. When mixing a fixative, check the osmolarity before adding the chemical fixative because the chemical fixative crosses membranes and does not contribute to the tonicity of the fixative. Most labs have a pH meter and most core tissue culture labs or microscopy labs have an osmometer. 

Recently, we prepared radioactively labeled erbstatin in the biosynthetic manner (7), and using it we assessed its stability in cell culture medium. Erbstatin was degraded about 50% within 30 min. On the other hand, the intracellular concentration in A431 cells reached half maximal in 2 hours. 

Although the platinum polymers share many properties with cis-DDP. one important difference between the two types of compounds has been uncovered and is indicated in Table 2. The biological activity of cis-DDP depends upon the age of the solution. A freshly prepared solution of cis-DDP in either dimethyl sulfoxide or cell culture medium is 100-fold more active than a solution which has been stored for several months. This loss of activity is presumably due to the reaction of cis-DDP... 

In this section, the application of the sensing mechanism described in Section 3.1 to an in vitro environmental model containing interfering cell culture medium is described. In this experiment the cell culture medium was used in place of deionized water, that is, 0.5 ml DMEM F-12 culmre media containing 1% fetal bovine serum (FBS) was mixed with 5.5 ml AuNPs stock solution to obtain a final AuNPs concentration of 2.25 X 10 NPs ml with pH 6.85. Additionally, to adjust the ion concentration, as was done previously, 100 pi of 10 mM FeCl3 at pH 2.90 was added into the mixture. Finally, 1 ml of 15 mM GSH at pH 3.44 was added to mediate the AuNPs. All optical characterizations were performed within 6 h of solution preparations. 

Prepare 1 10 dilutions ofvimses in microcentrifuge tubes by mixing 10 pL of PI virus with 90 pL ESF 921 insect cell culture medium (Expression Systems). 

Two identical cell cultures are prepared with the exception that one is grown in a medium containing specific, labeled ( N or C)... 

In an early report by Janes et al. [135], the interaction between DOX-loaded PEC nanoparticles (consisting of CHT and polyanions) and human melanoma cells was investigated with reference to the polyanion type and preparation protocol. Evidence was found that DOX-loaded PEC particles consisting of DS maintained the cytostatic activity with respect to free DOX, whereas DOX precomplexed with CHT before complexation with the polyanion showed slightly decreased activity (according to the MTT assay). Furthermore, CLSM studies revealed that DOX release did not take place in the cell culture medium, but that DOX was taken up into the melanoma cells by endocytosis while stUl bound to PEC particles and finally released intracellularly. 

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