Serum medium preparation

Dilute the peptide stock solution in Opti-MEM I Reduced Serum Medium to 500 pi in a microcentrifuge tube so that the weight ratio of peptide to siRNA (prepared in step 1) is 10 to 1 (see Notes 3 and 4). 

Serum Medium (as prepared in Subheading 3.1) to the cells with a final volume of 1 ml per well (each well contains 100 pmol siRNA) (see Note 6). 

Cationic liposomes composed of 3(3- [ N- (N N-dimethylaminoethane (carbamoyl] cholesterol (DC-Chol) and dioleoylphosphatidylethanolamine (DOPE) (DC-Chol/DOPE liposome, molar ratio, 1 1 or 3 2) prepared by the dry-film method have been often used as non-vtral gene delivery vectors. We have shown that a more efficient transfection in medium with serum was achieved using DC-Chol/DOPE liposomes (molar ratio, 1 2) than those (3 2), and preparation method by a modified ethanol injection than the dry-film. The most efficient DC-Chol/DOPE liposome for gene transfer was molar ratio (1 2) and prepared by a modified ethanol injection method. The enhanced transfection is related to an increase in the release of DNA in the cytoplasm by the large lipoplex during incubation in opti-MEM 1 reduced-serum medium (optiMEM), not to an increased cellular association with the lipoplex. Cationic liposomes rich in DOPE prepared by a modified ethanol injection method will help to improve the efficacy of liposome vector systems for gene delivery. 

Fig. 10 Release of cromolyn sodium (sodium cromoglycate) from human serum albumin microspheres prepared using a water-oil emulsion technique with 5% glutaraldehyde as cross-linking agent. Dissolution medium pH 7 phosphate buffer. (From Ref. 98.)... 

HeLa S3 (ATCC CCL-2.2) cells, a clonal derivative of the parent HeLa line (ATCC CCL-2), which are adapted to grow in suspension and therefore more suitable for large biomass production, are used for the preparation of human cell extract. The cells are maintained in suspension culture in Coming 850 cm2 Polystyrene Roller Bottles at 37° at a concentration of 3 to 6 x 105 cells/ml in Eagle s Minimum Essential Medium Joklik Modification (Sigma) supplemented with 10% Fetal Bovine Serum (Invitrogen) in the presence of 5% C02. 

There have been several studies that underscore the importance of unbound concentration in cell-based studies of receptor function. In a model study of the effect of plasma protein binding on the renal transport of organic anions using the expression of various organic anion transporters (OATPs) in Xenopus oocytes, the transport of ochratoxin A, methotrexate, and estrone sulfate was found to be strongly inhibited by the addition of human serum albumin to the culture medium [16]. Similarly, the addition of oq-acid glycoprotein was found to reverse the blockade of sodium-ion current by cocaine in a preparation of cardiac myocytes [17]. 

Prepare treatment medium containing various concentrations of test compound 19.7 ml of Eagle s medium (without serum) plus 300 pi of stock concentration of compound in a preferred solvent (e.g., water, ethanol, DMSO, etc.). The final concentration of solvent other than water should not exceed 1% v/v. Normally a range of 0-5000 pg ml-1 (final concentration) is covered. For a sparingly soluble compound, the highest concentration will be the lowest at which visible precipitation occurs. Similarly, if a compound has a marked effect on osmolality, concentrations should not be used that exceed 500 milliosmoles (mosm) per kg. In addition, a pH range of 6.5-7.5 should be maintained. 

Cells were grown for 24 h, growth medium was replaced for serum-free medium and the appropriate preparations were introduced, plates were illuminated with halogen lamp (about 45mW/cm2) and further incubated for 18 h in the darkness. Viability was assessed by Alamar Blue reduction. Results are presented as percent of control. 

The transfection efficiency and stability of some peptide/ siRNA complexes may be affected by the presence of serum. It is suggested to prepare the complexes in the absence of serum initially. Serum-supplemented culture medium can be used with complexes that show good transfection efficiency in the absence of serum. 

Brandley and Schnaar [140] immobilized a synthetic nonapeptide, Tyr- Ala-Val Thr-Gly Arg-Gly-Asp-Ser, on a polyacrylamide gel, which had been prepared by a ternary copolymerization of acrylamide, bisacrylamide and the acrylic ester of. V-hydroxysuccinimide. They reported that Balb/c 3T3 mouse fibroblast cells (in Hepes-buffered Dulbecco s modified Eagle medium) adhered readily to the peptide-derivatized surfaces, even in the absence of serum, although long-term cell growth required the presence of serum. It was noticed that reference nonapeptide. Tyr-Arg-Leu-Glu-Asp-Pro-Ala-Met-Trp, which has no RGD sequence, failed to promote cell-attachment. 

A combined in vivolin vitro system for chemical metabolites has been developed that demonstrates the role of metabolism in activating compounds. In this system, rats are treated with a chemical and serum is removed 1 h later and cultured with explanted embryos. This system has been used to demonstrate that metabolites of procarbazine are toxic to explanted embryos, but that procarbazine and a microsomal preparation are not toxic to explanted embryos grown in culture medium (Schmid et al., 1982). 

Feeder cells for fusion cultures (see Note 6) essential for fusions employing rat myelomas. Quickly thaw irradiated rat fibroblasts, prepared as described in Note 6, just before commencing the cell fusion. Add the cells to 10 mL of serum-free DMEM, centrifuge, and wash once in serum-free DMEM Resuspend feeders in HAT medium just before addition of the fusion mixture. Alternatively, use thymocytes from spleen donors (mouse). 

All solutions should be prepared in DMEM or other suitable growth medium containing 5% FCS (or newborn calf serum [NBS], which, if tested for non-toxicity, may be used as a cheaper alternative). If the effect of antibody binding on the behavior of the membrane protein is unknown, then all incubations should be carried out at 4°C (float plates on ice bath and precool diluents). Monolayers of cells vaiy widely m their adhesion to plastic, and also, they may roundup after prolonged incubation at 4°C owing to depolymenzation of microtubules (see Note 7). 

Prepare cell suspension from epithelial tissue by collagenase digestion resuspend cells in RPMI + 2% fetal calf serum at 106 cells/mL. Use this medium throughout... 

Prepare target cell suspension and dilute to 106 cells/mL in RPMI containing 2% fetal calf serum. Use this medium as diluent, and wash medium throughout. 

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