Neutral red dye

Primary antibodies against RUNX1 (#4334), RUNX2 (#8486), p27^Kip1 (#3698), and PARP (#9532) were purchased from Cell Signaling Technology (Danvers, MA, USA); antibodies against RUNX3 (ab40278), Cyclin D2 (ab207604), and tubulin (ab59680) were purchased from Abcam (Toronto, ON, Canada); antibodies against FLAG (F1804) and β-actin antibody (A5441) were purchased from Sigma–Aldrich (Oakville, ON, Canada); and the antibody against PEBP2β (CBFβ) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Secondary antibody IRDye 800CW conjugates of donkey anti-rabbit-IgG were purchased from LI-COR Biosciences (Lincoln, NE, USA). Neutral red dye was purchased from Sigma–Aldrich (Oakville, ON, Canada).

For generation of NR-DENV, C6/36 cells were infected with DENV-2 at 33°C for 1 h in FBS-free DMEM (DMEM-0) then incubated with DMEM containing 2% FBS (DMEM-2) containing 100 μg neutral red dye (Sigma) for 5 days in the dark. Following this incubation, the medium was harvested, cleared from cellular debris by low-speed centrifugation, aliquoted, and stored at −80°C. NR-DENV titers were measured in either dark or light-exposed conditions in Vero cells using a foci forming unit assay as described [54 (link)]. All experiments with NR-DENV were performed under semidark conditions, unless otherwise stated.
For experiments measuring NR-DENV infection, 293T cells were transfected with the indicated plasmids and ~ 48h post-transfection, cells were infected with NR-DENV (MOI = 5) for 2 h in the dark. At this time, cells were illuminated on a light box for 20 min. In parallel, monolayers were maintained in the dark to control for effects unrelated to entry or illuminated at 0hr post-infection to verify the light sensitivity of NR-DENV. Cells were then washed, collected by manual pipetting, and then transferred onto naive 293T cells. Cells were infected for approximately 48h, washed and infection levels assessed by RT-qPCR.

Neutral red dye (Sigma‐Aldrich) dissolved in cell culture medium was incubated with differentiating hBM‐MSC for 2 hours before fixation in 0.1% Calcium Chloride + 0.5% paraformaldehyde (both from Sigma‐Aldrich). Dye retained by hBM‐MSC was solubilized in 1% acetic acid + 50% ethanol (both from Sigma‐Aldrich). Quantification of solubilized Neutral Red was then performed on an absorbance spectrophotometer at 540 nm.

The neutral red cell viability assay was performed to determine the viable cell mass in HAP1-p32-wt, HAP1-p32-T130M and HAP1-mock cultures. 5 × 10³ cells per well were seeded into a 96-well microtiter plate and incubated at 37°C and 5% CO₂ for 96 h. To determine the effect of oxygen depletion, transfectants were cultivated under normoxia with 21% oxygen or under hypoxia in an incubator providing hypoxic conditions of 2% oxygen for 72 h.
After incubation, cells were stained using a neutral red dye (Sigma-Aldrich) diluted 1:100 in IMDM for 2 h, washed and destained with a solution consisting of 50% pure ethanol, 49% bidistilled water and 1% pure acetic acid to release the incorporated dye into the supernatant. To analyze the neutral red dye uptake, absorbance was measured at 540 nm against a background absorbance of 690 nm in a spectrophotometer.

The cytotoxicity of bacteria on RAW 264.7 cells was measured by a neutral red assay based on the description by Fotakis and Timbrell (2006) (link) with a few modifications. The RAW 264.7 cells were seeded into 96-well plates at a density of 10⁵ viable cells per well. On the following day, the RAW 264.7 cells were washed with PBS to remove the antibiotics, the B. adolescentis re-suspended in DMEM (Lonza) were added onto the cells (50 μl per well) and incubated for 1 h at room temperature. Afterward the inoculum was aspirated and fresh complete DMEM without antibiotics was added (100 μl per well). Plates were incubated at 37°C and 5% CO₂.
After 2 days incubation, the medium of the cells was changed to neutral red dye (Sigma–Aldrich, St. Louis, MO, USA, 100 μg/ml) dissolved in DMEM (100 μl per well) and incubated for another 2 h at 37°C and 5% CO₂. Cells were then washed with PBS and the addition of elution medium (EtOH/AcCOOH, 50%/1%, 100 μl per well) followed by gentle shaking for 10 min so that complete dissolution was achieved. The optical density was read at 540 nm (OD₅₄₀).

Synoviocytes were plated at a density of 10⁴ cells/cm² in 96-well plates in normal cultures, at 7.5⁴ cells/cm² in co-cultures with PBMCs were used in a ratio 1:5. After exposure or not to cytokines and Cd for 1, 5 and 8 days, cells were incubated for 150 min with neutral red dye (80 µg/ml 0.33%; Sigma-Aldrich, St. Louis, MO, USA) at pH 6.5 in serum free DMEM. Confluent HepaRG cells viability after Cd exposure was also assessed by neutral red assay with the same protocol.
Biopsies were minced in small pieces of similar size and put in 24 well plates in normal DMEM medium (Eurobio, Courtaboeuf, FR). Supernatants were collected at day 0 for IL-6 production normalization and biopsies were treated afterwards with cytokines and Cd in triplicates. Cell proliferation was measured by the area occupied by the cells on the plate after 5 days using ImageJ™ software. Supernatants were collected at day 2, 5 and 8 after Cd exposure.
IL-6 production was quantified in supernatants by ELISA (R&D system, San Diego, CA, USA).