Assays cell concentration

Tamplin et. al. (54) observed that V. cholerae and A. hydrophila cell extracts contained substances with TTX-like biological activity in tissue culture assay, counteracting the lethal effect of veratridine on ouabain-treated mouse neuroblastoma cells. Concentrations of TTX-like activity ranged from 5 to 100 ng/L of culture when compared to standard TTX. The same bacterial extracts also displaced radiolabelled STX from rat brain membrane sodium channel receptors and inhibited the compound action potential of frog sciatic nerve. However, the same extracts did not show TTX-like blocking events of sodium current when applied to rat sarcolemmal sodium channels in planar lipid bilayers. 

In cytotoxicity assays the concentration of the cytotoxic compound reaching the cytosol is estimated via its apparent toxicity. Cytotoxicity is expressed as the concentration that inhibits the growth of the MDR-expressing cells by 50% or 20% (known as IC50 or IC2o values, respectively), and indicates whether the secondary, co-administered compound is a substrate or a modulator of P-gp. The activity of the reversal agent is generally expressed as a fold reversion called the MDR ratio [36] ... 

Figure 3. Conversion of DBT in vitro by lysate prepared from IGTS8 cells with assay protein concentration of 5 mg/ml. The data represents concentrations of DBT (circles), HPBS (diamonds), and HBP (squares). The data fitting represents a consecutive reaction model with kx = 0.2/min, and k2 = 0.05/min. Figure extracted from Ref [53].

The light scatter assay may be used to determine absolute numbers of viable cells if flow cytometry data from cell suspensions of known concentration are used to construct a standard curve. For that purpose, cell concentrations should be determined in a series of graded, standard cell suspensions with the use of a Coulter counter. A plot of those standard concentrations versus the number of events (Hght scatter signals) acquired during a specified acquisition interval in the flow cytometer may then be used to interpolate cell concentrations for test samples that have been assayed by the light scatter procedure. 

Each reaction contains 1 x 10 neutrophils/mL of PBS. It is important to work with a cell density where cell clumping is not occnrring dnring the reaction. A density of 1 x 10 neutrophils/mL appears to be an optimal concentration for this assay. Cells should be kept at 0-4°C after isolation. 

Mammalian cells in culture are exposed to the test substance. Established cell lines are treated both with and without metabolic activation. Cells are incubated for an appropriate length of time, then rinsed, fixed, and dried. Slides are developed, stained, and exposed silver grains are counted. The endpoint of UDS is measured by determining the uptake of labeled nucleosides in cells that are not undergoing scheduled (S-phase) DNA synthesis. The most widely used technique is the determination of the uptake of H-TdR by autoradiography. Primary cultures (e.g., rat hepatocytes), human lymphocytes, or established cell lines (e.g., human diploid fibroblasts) may be used in the assay. Multiple concentrations of the test substance over a range adequate to define the response, should be used. 

Cytotoxicity Assays. Cells were plated into 60 mm diameter dishes at several different densities (250-500 cells per dish, three dishes per density) to obtain more than one set in which the number of surviving colonies ranged from 100 to 200. The cells were treated with increasing doses (20-160 pM) of butachlor, previously dissolved in ethanol, and the treated cells were left overnight. The next day, the old medium was replaced with fresh medium. The cells were incubated for 10 days, harvested, fixed with methanol, and stained with Giemsa s solution. Colonies containing more than 50 cells per colony were counted as survivors, and survival rates for treatment groups relative to the control were calculated. The concentrations of butachlor used to produce a survival rate of >20% were used in further experiments. 

Cells were transferred from an overnight liquid culture to fresh medium and grown overnight ( 18 h) on a gyrorotary shaker at room temperature. The cells were harvested by centrifugation (11,000 g, 10 min) and resuspended in sterile assay buffer (0.01 M phosphate buffer, Na salt, 0.26 M NaCl, pH 6.78 the pH prior to sterilization was 7.20). The cell concentration was adjusted to approximately 5 x 108/mL as judged by optical density of the solution. The actual numbers in dilutions of the cell suspensions were determined by direct counts. E. cloacae was grown as described, except that the M9 contained 0.5 g/L of NaCl. 

Enzyme source (see Note 4) Chinese hamster ovary cells (CHO-Kl, American Type Culture Collection) were transiently transfected via standard DEAE-dextran methods with an expression construct encoding the human ECE-lc protein. The medium was changed to serum-free medium the day after transfection. Two days after transfection, cells were harvested and a crude membrane fraction sedimented by ultracentrifugation (100,000g for 1 h). The pellet was suspended in assay buffer, aliquoted, and frozen at -70°C until assay. Protein concentration was determined by standard methods (e.g., Lowry or BCA). The equivalent of 3 pg total protein was used in each assay well. 

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... 

As a general rule, the most sensitive assays (using criteria 1 and 2 above) are AP NaOH. SRB, CVDE and NR, in that order. Both AP 2-h and MTT are the least sensitive, but they do have the greatest linear ranges, and this is an important consideration in some experiments where a very wide range of cell concentrations is expected. 

There are several assays available to quantitate cell death in bioreactors. The simplest assay uses Trypan blue dye exclusion from intact cells to determine hve and dead cell concentrations. Cells that have died and lysed, however, cannot be accounted for using this assay (see Chapter 2, section 2.2). 

In the neutral red (cell viability) and total protein (cell proliferation) assays, cells are treated with various concentrations of a test substance in petri or multiwell dishes after a period of exposure, the substance is washed out of the medium. (An analytical reagent is added in the case of protein measurements.) Neutral red is a supravital dye, which accumulates in the lysosomes of viable, uninjured cells, and it can be washed out of cells, which have been damaged. In the protein test, Kenacid blue is added and reacts with cellular protein. Controlled cells are dark blue killed cells are lighter colored. The IC50 (the concentration which inhibits by 50%) is determined the test can be rapidly performed with automation. However, materials must be solubilized into the aqueous cell media for analysis. For many test materials this will require large dilutions which eliminate properties of the materials which cause irritation. 

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