Aug 08, 2020
Version 2
- 1Universidade Federal do Rio de Janeiro
- L. P. Borba-Santos
External link: https://doi.org/10.1371/journal.pone.0240658
Document Citation: luanaborba 2020. Cytotoxicity assay using LLC-MK2 cell line. protocols.io https://dx.doi.org/10.17504/protocols.io.bjgbkjsn
Manuscript citation:
Borba-Santos LP, Vila T, Rozental S (2020) Identification of two potential inhibitors of Sporothrix brasiliensis and Sporothrix schenckii in the Pathogen Box collection. PLoS ONE 15(10): e0240658. doi: 10.1371/journal.pone.0240658
License: This is an open access document distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Created: August 07, 2020
Last Modified: August 08, 2020
Document Integer ID: 40163
Abstract
Protocol that described the cytotoxicity assay using a mammalian epithelial cell line (LLC-MK2; ATCC CCL-7).
Cytotoxicity assay using LLC-MK2 cell line
1. The mammalian epithelial cell line LLC-MK2 (ATCC CCL-7) was cultivated in RPMI 1640 medium1 supplemented with 2 mM L-glutamine and heat-inactivated 10% fetal bovine serum and buffered with sodium bicarbonate at pH 7.5;
2. Aliquots of 100 µl containing 2.5 x 105 cells/ml were added into flat-bottom 96-well microplates (TPP™) and incubated for 48 h at 37 ºC, in a 5% CO2 atmosphere, to obtain confluent monolayers of LLC-MK2 cells;
3.The supernatant was gently discarded, samples were washed in sterile PBS, and 100 µl of compounds previously diluted2 in RPMI 1640 medium were added on confluent monolayers;
4. Cells were treated for 48 h at 37 ºC, in a 5% CO2 atmosphere;
5.The supernatant was gently discarded and samples were washed in sterile PBS;
6. Cell viability was analyzed by the tetrazolium (XTT) reduction assay and 150 µl of XTT solution (1 mg/ml XTT1 and 1mM menadiona1 in PBS) were added in each well;
6.Microplates were incubated for 2 h at 37°C, in a 5% CO2 atmosphere, in the dark;
7.Microplates were centrifugated at 4000 rpm for 5 min and the supernatant was added into a new microplate;
8. Spectrophotometric readings at 492 nm were performed using a microtiter plate reader3;
9. The absorbance value for each well was subtracted from the value for the negative controls4 and inhibition of cell growth (I) relative to positive controls5 was calculated according to the following equation: I = 100 – (A x 100/C), where A is the absorbance of treated wells, and C is the absorbance of untreated control wells;
10. The concentration of compounds that elicited 50% cytotoxicity (CC50) was estimated by linear regression;
11. The diluent control containing 1% DMSO was included in experiments;
12. Experiments were performed in triplicate in two independent moments.
1Sigma Chemical Co., USA.
2 Stock solutions of compounds in dimethyl sulfoxide (DMSO) at 1 mM were diluted in RPMI 1640 medium supplemented with 2 mM L-glutamine to obtain concentrations of 0.1, 1, 2, 4, 5, 7, 8, 9, and 10 µM.
3EMax Plus, Molecular Devices, USA.
4 Wells without cells containing only RPMI media that were incubated in the same conditions of wells with cells.
5 Wells with untreated cells.