My work involves using pesticide formulations in vitro. However, even concentrations as low as 2.5% are proving to be highly cytotoxic and leading to cell-death. I have tried exposing the pesticide formulation for brief periods (~ 20 minutes) before changing the media to remove its traces (as recommended by the EPA protocol) but the cells do not survive (over 90% cell mortality rate)
It would be much appreciated if someone could recommend a protocol that would allow 50 % survivability .
Dear Shalini!
Please You lok at the pprotocol:
Article In vitro impact of five pesticides alone or in combination o...
2.1. Chemicals
The pesticides deltamethrin (CAS No. 52918-63-5), fenitrothion (CAS No. 122-14-5), fipronil (CAS No. 120068-37-3), lambda-cyhalothrin (CAS No. 91465-08-6), and teflubenzuron (CAS No. 83121-18-0) were purchased from Fluka (Riedel-de-Haën®, France).
2.3. Pesticide treatments
Pesticides concentrations assessed in our study were ranged from 0.1 to 100 μM. The chosen doses were based on the LMR values adapted to our in vitro study as follows:
2.4. Evaluation of cell viability by MTT assay
The effects of pesticides on Caco-2 cells proliferation and viability were monitored during a 10 days-period of exposure. Reduction of the permeant tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide (MTT) was monitored as described by Mosmann [24], with modifications. Briefly cells were seeded in 24-well plates (5 × 104 cells/well). After 20 h of incubation, the cells were treated with various concentrations (0.1–100 μM) of pesticides alone and in combination. At the end of the incubation time (3, 7 and 10 days of exposure), culture medium was discarded and the monolayer washed twice with PBS. Cells were then incubated with MTT (500 μl per well at a concentration of 0.5 mg/ml) in RPMI for 4 h at 37 °C. The reaction was stopped using 500 μl of a solution containing 10% SDS and 1 N NaOH at 37 °C. Then, 200 μl of formazan solution was transferred into 96-well plates and absorbances were measured by a microplate reader (Tecan, Lyon, France) at 570 and 690 nm. The relative cell viability was expressed as the ratio (%) of the absorbance in the experimental wells to that of the control wells (cells treated with DMSO). The EC50 (cytotoxic concentration for 50% cell death) was determined from the dose-response curve.
Article In vitro toxicity evalaution of low doses of pesticide in in...
Cell viability assay by MTT:
Cells (2 × 104) were seeded per well in 96-well plates and kept in CO2 incubator for 48 hrs. The treatments were performed with three type pesticides (50µL) by complete medium (200µL) and incubated for 24 hrs. Media was replaced by 3-(4,5- dimethylthiozolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) (5 mg/mL). The absorbance was recorded at 530 nm by using multiwell micro plate reader.
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