Revisiting the Resazurin-Based Sensing of Cellular Viability: Widening the Application Horizon

Abstract

Since 1991, the NAD(P)H-aided conversion of resazurin to fluorescent resorufin has been widely used to measure viability based on the metabolic activity in mammalian cell culture and primary cells. However, different research groups have used divergent assay protocols, scarcely reporting the systematic optimization of the assay. Here, we perform extensive studies to fine-tune the experimental protocols utilizing resazurin-based viability sensing. Specifically, we focus on (A) optimization of the assay dynamic range in individual cell lines for the correct measurement of cytostatic and cytotoxic properties of the compounds; (B) dependence of the dynamic range on the physical quantity detected (fluorescence intensity versus change of absorbance spectrum); (C) calibration of the assay for the correct interpretation of data measured in hypoxic conditions; and (D) possibilities for combining the resazurin assay with other methods including measurement of necrosis and apoptosis. We also demonstrate the enhanced precision and flexibility of the resazurin-based assay regarding the readout format and kinetic measurement mode as compared to the widely used analogous assay which utilizes tetrazolium dye MTT. The discussed assay optimization guidelines provide useful instructions for the beginners in the field and for the experienced scientists exploring new ways for measurement of cellular viability using resazurin.

Immune checkpoint inhibitors modulate the cytotoxic effect of chemotherapy in lung adenocarcinoma cells

“…The dose-response curves for chemotherapeutic agents in the presence or absence of therapeutic antibodies were performed in the 96-well format. HCC-44 or A549 cells (passage number below 30) were seeded in growth medium (RPMI-1640 or DMEM supplemented with 10% FBS) onto plate at a density of 2,000 cells or 3,500 cells per well, respectively [within the linear range of the method according to the preliminary experiments (16)]. After incubation for 24 h, the growth medium was exchanged, and dilution series of compounds in PBS or PBS supplemented with therapeutic antibodies were added onto the cells (1/10 of the growth medium volume).…”

Effects of Subtoxic Concentrations of Atrazine, Cypermethrin, and Vinclozolin on microRNA-Mediated PI3K/Akt/mTOR Signaling in SH-SY5Y Cells

“…Based on these results, EDCs were tested at 100 nM and 1 μΜ in the subsequent experiments. SH-SY5Y cells were induced to differentiate into mature neurons and the AlamarBlue HSTM test was used to assess cytotoxicity [ 28 ]. As revealed by the percentage of viability, the EDCs were not cytotoxic also in differentiated SH-SY5Y ( Figure 2 D).…”

“…As the differentiated SH-SY5Y cells detached more easily, the neuronal viability was evaluated using the AlamarBlue HSTM test by the conversion of resazurin to resorufin [ 28 ]. Briefly, differentiated SH-SY5Y cells were cultured in a 96 well plate at 2 × 10 3 cells/well and treated with VNZ, CYP and ATZ at the concentration of 100 nM or 1 µM for 48 h at 37 °C in 5% CO 2 .…”

Semisynthetic Sesquiterpene Lactones Generated by the Sensibility of Glaucolide B to Lewis and Brønsted–Lowry Acids and Bases: Cytotoxicity and Anti-Inflammatory Activities

“…To evaluate the potential antitumoral activity of the compounds, the MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2 H -tetrazolium bromide] method [ 75 ] was used to determine the cell viability of SK-MEL-28 (human melanoma cell line), NCI-H460 (human lung carcinoma cell line), SF295 (human glioblastoma cell line), and HUVEC (human umbilical vein endothelial cells) line. Cell viability analysis of murine macrophage J774A.1 was conducted using a resazurin-based cytotoxicity assay (Alamar blue ® ) [ 76 ]. All cell lines were acquired from the BCRJ (Rio de Janeiro Cell Bank, Federal University of Rio de Janeiro, Brazil).…”