Fish and Fishery Products Analysis by Saleena Mathew & Maya Raman & Manjusha Kalarikkathara Parameswaran & Dhanya Pulikkottil Rajan

Author:Saleena Mathew & Maya Raman & Manjusha Kalarikkathara Parameswaran & Dhanya Pulikkottil Rajan
Language: eng
Format: epub
ISBN: 9789813295742
Publisher: Springer Singapore

Fractionation of Gambierdiscus extracts – The MTX and CTX extracts should be resuspended in 0.5 ml of 30% acetonitrile (ACN). It is fractionated and eluted at 0.7 ml min−initially for 5 min at 5% acetonitrile (ACN)/0.1% formic acid followed by a linear gradient from 5 to 90% ACN/0.1% formic acid over 60 min. 70 × 1 min fractions should be collected. 25% (v/v) of these fractions should be freeze-dried and resuspended in 15 μl physiological salt solution containing 0.1% bovine serum albumin (BSA) just prior to FLIPR assay analysis.

Measurement of CTX- and MTX-like activity – Human-derived neuroblastoma cells, SH-SY5Y, express a range of voltage-gated sodium channel subtypes. Sensitive direct detection of MTX-induced calcium influx and indirect detection of CTX activity is possible by the use of these cells. It is done by measuring enhancement of veratridine-induced calcium influx. SH-SY5Y cells should be maintained at 37 °C/5% CO2 in RPMI media comprising 15% fetal bovine serum (FBS) and 2 mM L-glutamine. Cells should be routinely passaged at a 1:5 dilution every 3–5 days using 0.25% trypsin/EDTA. SH-SY5Y cells should be seeded at 120,000 cells/well in 40 μl of culture medium on black-walled 384-well imaging plates and cultured until 90–95% confluent monolayers are obtained. Cells are then loaded for 30 min at 37 °C with 20 μl of calcium-4 no-wash dye in PSS containing 0.1% BSA. The dye is absorbed into the cells’ cytoplasm during this 3-0min incubation period. Plates holding the loaded cells should be then transferred to the fluorescent plate reader with a 470–495 nM excitation filter and 515–575 nM emission filter. Camera gain and intensity should be adjusted to yield a minimum baseline fluorescence of 1000 arbitrary fluorescence units (AFU). After 10 s baseline measurement should be recorded. 10 μl of sample should be added to each well containing the loaded cells using injection manifold. Fluorescence should be recorded every second for 300 s thereafter.

Responses are normalized to baseline, to compute responses induced by MTX-like activity. The maximum increase in fluorescence for reads 10–300 should be determined. For P-CTX and CTX-like activity, the maximum increase in AFU over baseline (reads 300–310) should be determined for reads 310–600. By determining the peak AFU for each CTX fraction, results should be normalized. In the absence of a suitable standard, to establish the relative levels of MTX, results should be normalized to the Gambierdiscus/Fukuyoa extract that produced the largest increase in AFU (100% activity) of the samples tested. Each fraction from both MTX and CTX should be measured in triplicate.

Download

Copyright Disclaimer:
This site does not store any files on its server. We only index and link to content provided by other sites. Please contact the content providers to delete copyright contents if any and email us, we'll remove relevant links or contents immediately.