Trypan blue

The viability of macrophages after bacterial infection were determined by trypan blue exclusion and Lactate Dehydrogenase (LDH) assays. For trypan blue assay, equal volume of 0.4% of trypan blue (Sigma-Aldrich) was added to cell suspension and the number of viable cells enumerated. The LDH assay was carried out using the CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit (Promega) according to the manufacturer’s protocol.

As previously reported [33 (link)], Trypan Blue (Sigma-Aldrich) assay was used to evaluate the cell viability of both RPE-1 and U87 cells. Cells were seeded into cell culture plates at a concentration of 5 × 10⁵ cells/mL and kept for 24 h at 37 °C with 5% CO₂. Then, cells were treated with different concentrations of RDS 3337 (80, 320, 1280 nM) for an incubation time of 24, 48, or 72 h. Vehicle-treated cells or cells incubated with RDS 3337 were analyzed by Trypan Blue (Sigma-Aldrich) assay to assess cell viability. DMSO is the vehicle to dissolve the compound, and we consider cells with only DMSO as vehicle-treated cells. All experiments were carried out in quintuplicate.

The animals were anesthetized with a lethal dose (0.5 mL of 10% ketamine hydrochloride and 2% Xylazine solution) and PMN were collected from the peritoneum after injection of 3 mL of sterile PBS and further extraction of the whole PBS/exudate solution; 1 mL of this solution was placed on a Suta sedimentation chamber and then the slide was stained with May-Grunwald-Giemsa-Rosenfeld. Differential counting was performed with an optical microscope, in which granulocytes were distinguished from mononuclear cells. Cells were quantified using a hemocytometer and cell viability was assessed with 0.2% Trypan blue (Sigma). Regarding the subcutaneous route, PMN were collected 15 days after the infection of the mice. The animals were anesthetized with a lethal dose (0.5 mL of 10% ketamine hydrochloride and 2% Xylazine solution); cells were collected and placed in sterile tubes with the help of a sterile glass Pasteur pipette after skin flap procedures. The cells were transferred and stored in Falcon tubes containing RPMI (Sigma-Aldrich, St. Louis, MO, USA) with 10% Fetal Bovine Serum (FBS, Sigma), refrigerated (2–6°C), and quantified using a hemocytometer; cell viability was assessed with 0.2% Trypan blue (Sigma).

Cell death in N. benthamiana leaves was examined by using trypan blue staining. Agroinfiltrated leaves were harvested at 2.5 dpi and soaked in boiling trypan blue solution [10 mL lactic acid, 10 mL glycerol, 10 mL ddH₂O, 10 g phenol and 20 mg trypan blue (Sigma-Aldrich)] for 5 min and incubated for 3 h. Samples were then decolorized in 2.5 g/mL chloral hydrate solution to clear the background and photographed.

Myeloma cancer cells (RPMI8226 (ATCC^® CCL–155™)), promyelocytic leukemia cells (HL60 (ATCC^® CCL–240™)), and acute monocytic leukemia cells (THP1 (ATCC^® TIB–202™)) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). PBMCs were isolated from buffy coat purchased from the Central Blood Bank (Lodz, Poland). PBMCs were isolated with Histopaque 1077 (Sigma Aldrich, St. Louis, MO, USA) by density gradient centrifugation at 300× g for 30 min at 22 °C. The final concentration of lymphocytes was estimated by trypan blue (0.4%, Sigma Aldrich) exclusion assay. All investigated cells were suspended in RPMI 1640 medium supplemented with 1% phytohemaglutinin (only in PBMC growth medium), 10% fetal bovine serum, penicillin (10 U/mL), and streptomycin (50 μg/mL) in standard conditions: 37 °C, 100% humidity, and an atmosphere of 5% CO₂ and 95% air. Cell viability was systematically controlled using trypan blue (0.4%, Sigma). In all experiments, cells in the logarithmic phase of growth were used when their viability was above 95%.