RPMI medium

Count the CLL cells, centrifuge at 200 for 5 min at room temperature (RT), and resuspend in fresh complete RPMI media at 2 x lO " cells/ml. 

RPMI media and fetal calf serum (Gibco-BRL). 

RPMI media (or any cell-specific media) without serum. 

The rhizomes of Alpinia qfficinarum were purchased from Nuherbs Co. (Oakland, CA). Du-145 prostate tumor cells were obtained from the American Type Culture Collection (ATCC). Cells were maintained at 37°C in an atmosphere of 5% CO2 and grown in Roswell Park Memorial Institute (RPMI) media 1640 (GIBCO/BRL) with 10% fetal bovine serum and with 5% penicillin and streptomycin. Cells ere routinely checked and foimd to be free of contamination by mycoplasma. 

Primary CLL cells purified from peripheral blood of volunteer patients (see Note 3) CLL cells are purified by Ficoll gradient centrifugation, by negative selection, or by a combination of both (see Note 4). CLL cells can be used immediately or frozen in 90% heat/inactivated FBS supplemented with 10% DMSO and stored at -80°C or in liquid nitrogen for short-and long-term storage, respectively. CLL cells are cultured in complete RPMI medium and maintained in a humidified atmosphere of 5% CO at 37°C (see Note 5). 

Examine the cells under the microscope to ensure that all the fibroblasts are attached to the tissue culture dishes (Fig. la). Wash the layer of fibroblasts twice with 5 ml of pre-warmed PBS. Seed 2x10 CLL cells (ratio 10 1 10 CLL cells per NIH3T3 cell Fig. lb) on WT and CD154 N1H3T3 in 5 ml complete RPMI medium supplemented with 10 ng/ml of lL-4 (only in CD154 NIH3T3-coated tissue culture dishes) and incubate for 24 h in a humidified atmosphere of 5% CO at 37°C (see Notes 7 and 8). 

C RPMI medium, 5% ECS (v/v), 0.35ml/100ml 2-mercapto-ethanol, lOU/lOOml streptomycin, lOU/lOOml penicillin, 29 mg/100 ml L-glutamine... 

Cells were grown in RPMI medium plus 5 % fetal bovine serum in 1 mL total volume as described in Materials and Methods. At 24-hr intervals the cells were counted in a Coulter counter. Control cells 0, cells from cultures containing 1000 lU/mL mouse fibroblast interferon , cells from cultures containing bovine brain gangliosides at a concentration corresponding to 35 p.M sialic acid , cells from cultures containing both interferon (1000 IU/mL) and gangliosides (35 p.M sialic acid). 

RPMI medium 1640 (Gibco, Invitrogen, Carlsbad, CA) supplemented with 25% (v/v) of human serum (Fisher BioReagents, Fisher Scientific, Pittsburg, PA) (see Note 2). 

The supernatant was decanted, the pellet resuspended, and 1 mL of fusion media containing 40% polyethylene glycol (PEG), 10% DMSO, in RPMI medium (no FCS) was added slowly over 30 s with continuous gentle tapping to suspend the cells. 

The mixture was allowed to stand for 2 min, then 0.25 mL of 5% DMSO, in RPMI medium (no FCS) was then added every 15 s for a total of 10 min. Then PBS was added to a total of 50 mL, and the tubes were spun at room temperature. 

Figure 4.6.1 Equilibrium concentration (mM) of dissolved oxygen in RPMI medium at different oxygen partial pressures (mmHg). Based on data of Oiler et al. (1989) by permission of Journal of Cell Science.

The spleen was removed and cut into several small pieces and was gently forced through a 400 mesh stainless steel screen into a Petri dish which also contained RPMI medium. The cell suspension was transferred to a sterile centrifuge tube and any large tissue aggregates were removed by the sedimentation procedure described by Shortman et al. (lfi). The suspension was centrifuged (200 x g) for 10 minutes and the cell pellet was resuspended in fresh medium. 

Cells in trypan blue viability stain were enumerated microscopically. The spleen cells were mixed with an equal number of SP/2.0 myeloma cells in the semi-log growth phase in RPMI medium. The cell mixture was centrifuged (200 x g) for 10 minutes and the cell pellet was suspended in 1 mL of polyethylene glycol (3 000 - 4 000 mol. wt. range) at 37 °C. The suspension was mixed continuously for one minute, followed by the addition of 1 mL of RPMI medium and another one minute of continuous mixing. An additional 9 mL of RPMI medium... 

The culture showing the best results from an EIA assessment designed to detect the presence of picloram specific antibodies was selected for the limiting dilution procedure to acheive the clonality of the hybridoma cells. A dilution was calculated to yield one cell per well of a 96 well microtitration plate. The wells of the plate were checked daily for the presence of a single colony. Once a colony was visible, it was fed with 125 uL of RPMI medium. 

Grow 2.2.15 cells (7) (or cells of another HBV producer line) to confluence in RPMI medium plus 10% fetal bovine serum (see Note 3). 

Resuspend in a 1 1 solution of skim milk stock and RPMI medium containing 10% FBS, so that the final cell density is 107 cells/mL. 

Interleukin-2 (IL-2). Used as solution of 200 U/mL in RPMI medium supplemented with 15% heat-inactivated FCS, 2 mM L-glutamine, 0.1% sodium bicarbonate, and 20 pg/ml. gentamycin. This solution is stored at -20°C. 

I-labelled siRNA solution 4.8 pig/ml total siRNA (GFP-22 siRNA, Qiagen) comprising 2 x 10 CPM/ml I-labelled siRNA from Step 3.3 in RPMI medium without supplements, or whatever solvent of interest. 

Multiplicities of infection (MOI) of 10 1 and 2x10 1 (Salmonella THP-1 cells) were used. Salmonella cells were harvested at 3500 rpm for 8 min, washed 3 times in PBS and resuspended in 200 pL fresh antibiotic free RPMI medium. Bacteria and THP-1 cells were incubated together for 40 min at 37 C and were then decanted into sterile tubes and washed twice in pre-warmed PBS. Colistin was added at 50 pg/mL to inactivate extracellular bacteria and 200 pL of the cultures were placed into a black, clear-bottomed 96 well-plate. 0.1% saponin was added at time 180 min where appropriate. Imx and lux cultures of S. Typhimurium DT104 were used as controls. Bioluminescence was measured over 24 hours in an automated luminometer (Anthos) at 37 °C. 

Fish test sample working solutions are adjusted to 2 g TE/mL in RPMI medium and then serially diluted (8x) in RPMI medium in separate vials. 10 pL from each dilution is added in triplicate to the 24 +OA and 24 -OA wells. Similarly, CTX-1 standard (0.5 ng/mL) is serially diluted (8x) and 10 pL from each dilution added in triplicate to 24 +OA and 24 -OA wells of the toxin standard plate. The 96-well plate cultures are then incubated 22-24 h. The treatment medium is removed at 22-24 h by hand-flick, replaced with (3-[4,5 dimethylthiazol-2-yl]-2,5-diphenyltetrazolium) (MTT) solution (0.83 mg/mL RPMI medium 60 pL/well), and incubated for 30 min. The MTT solution is removed from the plates after 30 min by hand-flick and replaced with DMSO (100 pL/well). The plates are agitated gently side-to-side to disperse the formazan reduction product of MTT before measuring absorbance at 570 nm (reference wavelength of 630 nm) using a microplate reader. 

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.

Complete media RPMI-1640 medium (stored at 4°C, wrapped in aluminum foil to protect it from light) supplemented with a 100 U/mL penicillin, 100 p.g /mL streptomycin mixture, 4 mM L-glutamine and 7.5% heat-inactivated FCS (aU from BioSource International, Camarillo, CA). Allow the RPMI medium and the reagents to reach 37°C in a warm water bath before the reagents are added to the media. This complete media can be stored at 4°C for a maximum of 2 wk. 

ConA 5 mg powder stock of ConA lectin from Canavalia ensiformis, 5 mg powder vial (Sigma, Saint Louis, MO). Con was reconstituted in 1 mL RPMI medium (5 mg/mL stock), aliquoted into 50-[iL fractions and stored at -20°C. 

Carefully rinse the cell suspension in the filter screen with 15 mL of RPMI medium to harvest all the spleen cells. 

Flow Cytometric Analysis of Intracellular Ca. PC-3 cells (2 X 10 ) were loaded with 1 pM indo-1 AM in serum-free RPMI medium for 30 min at 37°C, washed, and resuspended in 1 mL of the same medium. Indo-1 fluorescence was monitored with a MoFlo instrument (Cytomation) using a Coherent 1-90 argon laser tuned to ultraviolet for indo-1 fluorescence excitation. Intracellular Ca " was measured by comparing the ratio of indo-1 emission at 405 and 520 nm with excitation at 350 nm as described previously (66). Analysis was performed using Cyclops Summit software. 

Fig. 4. Wortmannin-induced apoptosis in LNCaP cells. (A) Dose- and time-dependent effect of wortmannin on cell viability of LNCaP cells in RPMI medium supplemented with 0.1% fetal bovine serum. The data represent the mean of three independent determinations. (B) Dose- and time-dependent effect of wortmannin on DNA fragmentation. The first lane is a 100-bp DNA ladder. (C) Wortmannin (0.5 pM) inhibits Akt phosphorylation in a time-dependent manner in LNCaP cells. In contrast, wortmannin was not effective against PC-3 cells. Source Reprinted from Reference 74 with permission from Elsevier.

RPMI medium with Glutamax I, supplemented with 10% FBS (Invitrogen). Store at 4 °C. 

Passage confluent Vero cells in a single T-150 flask 1 12 into 12 T-150 flasks. This split will provide confluent monolayers in new flasks for C. burnetii infection in 24 8 h. Grow cells in RPMI medium with 10% FBS at 37 °C in 5% CO2. 

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. 

Chinese hamster ovary (CHO) cells were maintained as monolayers in McCoy s 5A medium supplemented with 10% fetal calf serum and antibiotics. Normal human lymphoid cells, WIL-2 were maintained as suspension cultures in RPMI medium containing fetal calf serum and antibiotics. 

CLL Cells isolated from peripheral blood by density gradient centrifugation were cultured for 12 hr in the absence or presence of 1 iM CdA in nicotinamide-free RPMI medium, supplemented with L-glutamine 2 mM and 20% autologous plasma. Neutralized perchloric acid extracts were analyzed for NAD content by an enzyme cycling assay, and for ATP content by HPLC. 

HeLa cells are grown on 10 cm tissue culture dishes for 2 days to 90% confluence in RPMI medium containing 10 mM Hepes, pH 7.3, and 10% FBS. After rinsing cells once with warm complete MEM medium, 10 ml of warm complete MEM medium is added and the dishes are incubated for 1 h at 37° in a CO2 incubator. 

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