RPMI medium preparation

During the incubation time, fill each of the remaining wells of columns 1, 4, 7, and 10 with 180 pi of serum- and supplement-free medium (RPMI 1640). Prepare a 1 1 dilution series, when the 15 min incubation time is over, as follows transfer 180pl, from each of Al, A4, A7, and AlO to Bl, B4, B7, and BIO, respectively, using a multichannel pipette, then mix, transfer 180 pi from the respective wells in row B to row C and so on down to row D or further to cover the desired nucleic acid dose range per well. 

Fig. 3. Preparing a coverslip for live cell microscopy, (a) Dissolve 1 mg of bovine fibronectin In sterile water. After 1 h, add 4 ml of PBS and store 200 pg/ml fibronectin solution at 4°C. (b) Remove gaskets trom plastic permanox 8-well chamber. Cut epoxy mold squares and stick to No. 1.5 gold seal cover glass. Add 125 pi ot tibronectin, let sit for 1 h, rinse once with RPMI culture medium, and store In RPMI medium until ready to Image.

To prepare the differentiated HL-60 cells 2x10, HL-60 cells are inoculated into 10 ml antibiotic-free RPMI medium supplemented with 10% FBS and 1.3% endotoxin-low DMSO is added. Cells are incubated at 37 ° C, 5% CO2 for 5—7 days. The morphology of cells will change during the process of differentiation from a perfecdy round shape to a randomly irregular shape with multiple protrusions on the cell membrane. 

A comparison of the results above suggests that subtle differences in experimental conditions could have pronounced effects on the apparent requirements for T cell activation. Lessard and Dupuis [12] reported that the effects of retinol and RA on T cell blastogenesis induced by Concanavalin A differed markedly depending on the preparation of the retinoid-containing medium added to cells. Blastogenesis was reduced when retinol was first diluted in RPMI medium and then added to serum-containing medium, but in contrast was increased when retinol was first diluted in fetal calf serum. The association of retinoids with serum proteins may be critical, and/or the effective concentrations or stability of retinoids may differ importantly depending on preparation. In any case, these disparate results illuminate the importance of mul-... 

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. 

Media basal RPMI 1640 medium requires supplementation with sodium pyruvate, L-glutamine, and penicillin/streptomycin before use. The supplements are supplied as concentrates of 50X or 100X, and the appropriate amounts should be added. Standard tissue culture media for hybridoma production contain 5%, 10%, or 15% fetal bovine serum (FBS see Note 6). Sufficient quantities should be prepared in advance and a sterility check should be performed on them prior to use. 

The method contains three stages including first establishing a cell line followed by test chemical exposure and finally evaluated for expression of cell surface markers. To establish a cell line, human leukocyte preparations are attained from a plasma distributor. The leukocyte preparations as described by Ryan et al. (2004), are diluted with an equal part of complete medium (RPMI 1640 containing 1 x L-glutamine, 1 x penicillin-streptomycin-neomycin antibiotic mixture), 30 p,2-mercaptoethanol and 10 % heat inactivated fetal bovine serum. The diluted preparation is layered onto a Ficoll-Paque gradient to... 

Candida albicans cells were prepared and used immediately to determine the minimum inhibitory concentration (MIC). RPMI-1640 was used as medium with L-glutamine buffered to pH 7 with 0.15 M 3-(Ar-morpholino)propane sulphonic acid solution. Candida albicans cells, 1.5 x 103 cells/ml, were added to the wells of a 96-well plate containing RPMI-1640 and treated with selected experimental... 

The mixed lymphocyte reaction (MLR) test was performed in 96-well flat-bottomed microtiter plates. Selected experimental agents were prepared as 10 mM stock solution in DMSO and a 50-fold dilution of this was prepared in RPMI. Serial dilutions were prepared from this subsequent solution and 10 xl of the diluted stock was added to the well to give concentrations in the assay starting at 9.5 xm. In each well was placed 1.5 x 105 donor cells to give a final volume of 0.2 ml RPMI 1640 medium supplemented with 10% human serum, 2mM L-glutamine, and penicillin/streptomycin. Cells were incubated at 37°C in a humidified atmosphere containing 5% carbon dioxide for 120 hours. 3H-Thymidine (0.5 xCi) was added in the final 6 hours of incubation and cell radioactivity levels determined, which were indicative of T-cell proliferation. 

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...

Cell lines usually cannot be successfully propagated in various synthetic media without supplemental (and, unfortunately, poorly characterized) serum components. This requirement for supplemental protein materials probably reflects—at least initially—the need for adsorbable proteinaceous constituents which will spontaneously accumulate at and favorably modify (for cell adhesion and propagation) the surfaces of the culture containers. There was essentially no spontaneous fllm adsorption from freshly prepared RPMI 1630 culture medium without... 

Resuspend virus pellets in 8 mL RPMI-1640 medium with 1.5% FCS. This virus preparation represents a 250-fold concentration. 

Fig. 2. Effect of the charge ratio (+/-) of ML (DC-Chol/DOPE=1 2) to plasmid DNA on size of lipoplexes formed by direct mixing which was left at room temperature for 15 min and incubated with RPMI-1640 medium for further 15 min. The lipoplex at various charge ratios (+/-) of DC-Chol to DNA were prepared by addition of each liposome preparation contained 0.9 mM DC-Chol concentration (1.67,10,16.7 Lforthe charge ratio (+/-) of 0.5,3 and 5) to 1 p g of DNA in 5pL of water...

For the competition assays, AR42J cells were seeded at a density about 800 000 cells per well on six-well plates and incubated for 24-48 h. Cells were then washed twice with RPMI-1640 medium supplemented with 10% FCS and antibiotics. The cells were supplied with fresh medium, and the radiolabelled preparation, corresponding roughly to 200 fmol peptide in 150 gL PBS/0.5% BSA buffer, was added. The competitor (Sandostatin) was added in ten concentrations (0.01, 0.1, 1.0, 3.0,10, 30,100, 300,1 000,10 OOOnM) in a total volume of 150 gL of PBS/0.5% BSA buffer. The cells were incubated for 2 h at... 

Preparation of ali-trans retinoic acid and methacrylate solutions. Stock dimethyl sulfoxide (DMSO) solutions of UDMA (from 27.5 mmol/L to 220 mmol/L), BDDMA ( 0.2 mol/L and 0.4 mol/L) and ATRA (1.00 mmol/L) were prepared immediately before use. As a general procedure, one of the above-mentioned solutions (1.0 pL) was added to 200.000 HL-60 cells in 1.0 mL of RPMI 1640 medium. 

Human Burkitt Lymphoma ( Raji ) cells were obtained frozen from the American Type Culture Collection thawed, and cultured, in RPMI containing 10% Fetal Bovine Serum. The polymers for microencapsulation were dissolved in distilled water with pH adjustment to 7.4, and dialysed extensively against distilled water through a 104 Da cut off dialysis bag. The polymers were then pH adjusted to 7.4 and freeze dried to constant weight. Final preparation for the microencapsulation process was limited to dissolution in appropriate medium and filtration through a 0.4 pm cartridge. 

RPMI 1640 medium (Invitfogen Life Technologies, Carlsbad, CA) Prepare as described under Subheading 2.1. 

The differentiated CXCR2-HL-60 cells are washed and resuspended with serum-free RPMI-1604 medium at a concentration of 4 X 10 cells/ ml. The prepared cells are injected into the device through the loading channel and seed the cells for 5 min at 37 °C and 5% CO2. 

Meier and coworkers have prepared a range of substituted benzyl esters (X = 4-Me, H, 4-Cl, 4-CN or 3-NO2) of the 5, 5 -nucleotide dimer of AZT (73) [90]. The triesters hydrolyzed cleanly to the nucleotide dimers in either phosphate buffer (pH 7.5) or RPMI culture medium/10% heat-inactivated foetal calf serum, at 37°C. The rate was dependent on the benzyl substituent, with half-lives ranging from 22 min (4-Me) to 9 days (3-NO2) in the RPMI culture medium. The triesters showed activity comparable with AZT against HIV-1 and HIV-2 in CEM cells. However, the triesters were not active against HIV-2 in a thymidine kinase-deficient cell line, suggesting the release of AZT rather than its 5 -monophosphate. 

Preparation of cell extracts. MOLT 4F is a human T-cell line established from a patient with acute lymphocytic leukemia (13) and has since been maintained in our laboratory. Characteristically, MOLT 4F cells form rosettes with sheep red blood cells (E-rosettes). They do not synthesize immunoglobulin and grow as a single cell suspension. RPMI 1788 is a human B-cell line established from the peripheral blood of a healthy volunteer. These cells produce immunoglobulin, have complement receptors and grow as clusters in suspension culture. Characteristics of these cell lines have recently been summarized by Moore (14). These cells were maintained in medium RPMI 1640 with 10% fetal calf serum and penicillin and streptomycin. The viability of the cells measured by a trypan blue... 

All animal treatment in this study conformed to the Guidelines for the care and use of experimental animals, in Rokkodai Campus, Kobe University . Mononuclear cells were prepared from male Wistar rats (7 to 8 weeks old Japan SLC, Shizuoka, Japan) and cultured at 1x10 cells/ml in RPMI 1640 medium (Nissui Pharmaceutical, Tokyo, Japan) containing 5% fetal bovine serum (Dainippon Pharmaceutical, Tokyo, Japan), 1 mM glutamine, and Img/ml kanamycin. 

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