Cell culture assay plates

In initial range-finding experiments, it is often practical to do one-factor-at-a-time experiments (4), followed by factorial screening designs using many factors (4,7), each at two levels. As we are interested in the properties of the concentration-response curve (particularly the difference between the asymptotes, the steepness of the responsive region, and the variation around the curve), it is important to quickly move to study the full curve. For cell-culture assays performed in 96-well plates, an effective approach is to assign... 

Llewellyn, L.E. et al., Measurement of paralytic shellfish toxins in moUuscan extracts comparison of the microtitre plate saxiphilin and sodium channel radioreceptor assays with mouse bioassay, HPLC analysis and a commercially available cell culture assay, EoodAddit Contam 18, 970, 2001. 

Tissue Culture Assay. Kogure et al. (48) report a novel tissue culture assay for detecting several types of sodium channel blockers. The mouse neuroblastoma cell line ATCC CCL 131 is grown in RPMI 1640 supplemented with 13.5% fetal bovine serum and 100 pg/ml gentamycin, in an atmosphere of 5% C0 95% air at 37 C. Ninety-six well plates are seeded with 1 x 10 cells in 200 pi of medium containing 1 mM ouabain and 0.075 mM veratridine. Veratridine and ouabain cause neuroblastoma cells to round-up and die. In the presence of sodium channel blockers (e.g., TTXs or STXs), the lethal action of veratridine is obviated and cells retain normal morphology and viability. An important feature of this assay is that a positive test for sodium channel blockers results in normal cell viability. Since bacterial extracts can contain cytotoxic components, this assay offers an advantage over tests that use cell death as an endpoint. The minimum detectable level of TTX is approximately 3 nM, or approximately 1/1000 mouse unit. 

For higher throughput applications, injection-molded plastic microtiter plates have served as the formats of choice for automated assay development. Thermoplastics such as polystyrene, polycarbonate, and polypropylene are used for a variety of purposes including storage and assay plates, lids, pipette tips, and Eppendorf PCR tubes. Polystyrene plates are used for cell culture and ELISAs. Polycarbonate reagent bottles are popular, while polypropylene storage plates and PCR tubes are standards. 

For the electrophysiological assay, BCjHl cells that express the muscle-type nAChR are plated in cell culture dishes. 

Tumor Spheroid-Based Migration Assay 1. Appropriate culture medium for the specific tumor cell line(s) under test. 2. 96-well flat-bottomed tissue culture-treated plates. 

Transfer the plate to a cell culture incubator. Four days later, visually confirm that the EC monolayer is confluent and proceed with the migration assay. 

The choice of cell culture method for cytotoxicity studies for a certain substance depends on the target cells and assay duration. Organ, spheroids, suspension cells, flask surface, and multi-well plate monolayers, as well as agar surface cultures can be employed. 

Activity Determination (Day 6) The cells are harvested for the evaluation of enzyme activities. This can be performed in situ via the incubation of the cells with the enzyme substrates (Table 2). Activity can also be determined from the cell homogenates or microsomal fractions of the cells. The in situ method is the most efficient, and would allow one to use 24-well or 96-well plates. The microsomal method would require the use of larger cell culture plates (e.g. 100-mmdiameterplates). DME substrates used in the hepatocyte enzyme induction assay are shown in Table 4. 

Measuring multiple parameters can be very useful for characterization of a cell culture model system during initial assay development and during subsequent optimization or miniaturization to a microwell plate format. The use of multiplexed internal controls also can be useful to identify false hits. 

Another approach to overcome assay chemistry incompatibility is splitting a sample into two different containers. If the cell population releases a marker into the surrounding environment, a small aliquot of culture medium can be sampled and moved to a different assay plate to segregate assay chemistries. Figure 6.11 shows the measurement of lactate dehydrogenase released from a... 

EC monolayers obtained after growth on either collagen-coated glass coverslips, or tissue culture multiwell plates as previously described (Sections 2.3.3.1 and 2.3.3.2), are rinsed several times in serum Iree medium, and then incubated 1 h at 37°C with assay medium (usually serum free culture medium supplemented with 0.5-1% BSA), before they are used as substrates for the adhesion assay. Tumor cells are detached from culture dishes either by EDTA or by trypsin treatment. In the latter case, trypsinized cells should be allowed to recover from the enzyme treatment in their growth medium for 30 min at 37°C. After rinsing in serum-free medium, labeled tumor cells are resuspended at the desired concentration in the assay medium and plated onto the EC monolayer at a cell concentration ranging between 10 and 2 x 10 /cm. Incubation is... 

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