Specific detection of two voltage gated sodium channel activators, pacific ciguatoxin (P-CTX3C) and brevetoxin (PbTx3) and two inhibitors, saxitoxin (STX) and decarbamoylsaxitoxin (dc-STX) was achieved, with EC50 values of 1 1.7 0.35 pg/mL, 5.8 0.9 ng/mL, 3 0.5 ng/mL and 15.8 3 ng/mL, respectively. Intra and inter-assays comparisons of viability controls, LOD, LOQ and toxicity in fish samples gave coefficients of variation (CVs) ranging from 3% to 29%. This improved test adaptable to either high throughput screening or composite toxicity estimation is a useful starting point for a standardization of the CBA-N2a in the field NBTGR of marine toxin detection. = 10 counts for each point. Coefficients of variation (CV) ranged from 10.8% to 26.3%. The linearization of the log phase of the growth curve was defined by the following equation: Y = 0.0578X + 7.7948 (r2 = 0.9974) (1) in which X is the culture time (hours) and Y is the Ln-transformed cell number. Based on this equation, it was concluded that the N2a growth curve was characterized by a 9.8 h lag phase (Y = Ln (4288)) and that the cell number increased by two-fold after an additional 12 h. Moreover, a cell seeding density of 50,000 10,000 cells/well allowed reaching a maximum cell density of 100,000 cells/well after 22 h culture time. For more convenience, a culture time of 26 h post-seeding was selected in all further experiments. NBTGR Next, the N2a cell initial viability after 26 h growth was compared at ten different cell seeding densities ranging from 10,000 to 100,000 cells/well and in two culture conditions (5% and 10% FBS growth medium) (Figure 2). Results showed that (i) the highest absorbance value is consistently obtained at a cell seeding density around the maximum number of cells supported by microplate wells, and (ii) absorbance values increase in proportion with MTT incubation times (Figure 2). For instance, the maximum absorbance value measured after 26 h culture time and 45 min ERK1 MTT incubation time was 1.4 while selecting a cell seeding density of 50,000 10,000 cells/well (vertical dotted lines, Figure 2) allowed to reach absorbance values comprised between 1 and 1.25 (horizontal dotted lines, Figure 2). This absorbance range was considered optimal when the detection of a decrease in cell viability is sought. Finally, no differences were observed when using either 5% or 10% FBS growth medium, which indicates possible saving opportunities on this expensive reagent at this step of the CBA-N2a. Open in a separate window Figure 2 Initial viability of N2a cells observed in 96-well microplates after 26 h growth in 5% FBS (full line) and 10% FBS (dotted line) culture medium, at different cell seeding density. Six distinct MTT incubation times were also tested: 15 min (blue); 25 min (green); 35 min (orange); 45 min (black); 55 min (pink); 65 min (red). Data represent the mean SD of one microplate (N2a cells at 536 P), each point tested in six wells. Mean CVs were <3%. Absorbance values were measured at 570 nm via the MTT assay. Based on these results, all further CBA-N2a experiments were conducted as follows: Implement cell layer in microplates using a cell seeding density of 50,000 10,000 cells/well in 200 L of a 5% FBS culture medium, in order to reach a maximum cell density of 100,000 20,000 cells/well after 22 h of culture. Conduct the MTT assay at an incubation time of 45 min, in order to reach an absorbance value 1.0 that is used NBTGR to define N2a initial viability. For each experiment, dedicate a separate microplate to measure the N2a cell initial viability after 26 h of growth defined as the Reference Cell Viability control (RCV control). 2.2. Characterization of N2a Cell Final Viability The second step of CBA-N2a is the exposure of N2a cells to VGSC activators or inhibitors, in OV? or OV+ conditions. Following an additional culture time of 19 h overnight, the final viability of N2a cells was assessed as previously described. 2.2.1. N2a Cell Final Viability.
Specific detection of two voltage gated sodium channel activators, pacific ciguatoxin (P-CTX3C) and brevetoxin (PbTx3) and two inhibitors, saxitoxin (STX) and decarbamoylsaxitoxin (dc-STX) was achieved, with EC50 values of 1 1
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