Trypan blue

For assessing MSN uptake, RBE4 cells grown on Ibidi dishes were incubated with MSNs in serum-free medium at 20 μg/ml for 24 hours, washed in PBS, fixed, incubated with Trypan blue (Sigma) at a concentration of 200 μg/ml in PBS for 7 minutes for quenching extracellular fluorescence, and mounted with SlowFade mounting medium (Thermo Scientific). Cells were visualized using a Leica SP8 confocal microscope (Leica Microsystems).
For visual evaluation of nanoparticle toxicity in transport experiments, MDCK II cells on the permeable supports from transport studies (after the final TER measurement) were washed twice in prewarmed PBS, incubated with Trypan blue at a concentration of 200 μg/ml in PBS for 7 minutes and then washed twice in PBS. The membranes were then removed and the cells observed using a Leica DM2500 B light microscope (Leica Microsystems).

A trypan blue assay (0.1%) was used to determine cell death [48 (link)]. MDA-MB-231 and MCF-7 cells were plated into 35 mm falcon tissue culture dishes (Thermo Fisher Scientific Nunc A/S, Roskilde, Denmark) with a 5 × 10⁴ cells/mL density and allowed to incubate overnight to settle before the application of the treatment. Bacterial cells were normalized to an OD₆₀₀ of 1. One hundred microliters from the prepared suspensions was added onto the confluent layer of breast cancer cells at a multiplicity of infection (MOI) of 1000:1 [49 (link)] in 6-well plates and incubated for an hour at 37 °C, 5% CO₂ and 100% relative humidity. Control cells received LB (vehicle control) only. Untreated breast cancer cells received only cell culture media (DMEM). Cell culture medium ± bacterial culture or control was placed with 0.1% trypan blue (Gibco by Life Technologies, Grand Island, NY, USA) in DMEM and incubated at 37 °C for 10 min. The trypan blue solution was then replaced with 1 mL of fresh culture medium, the cells were viewed at 20× magnification under an inverted microscope (Leica Microsystems Ltd. CH-9435, Heerbrug, Germany) and the percentage of alive cells ((unstained/stained) × 100) was calculated from 20 representative areas [47 (link),48 (link)].

The viability of the probiotic mix of Bifidobacterium lactis (1.75 BU/g), Lactobacillus acidophillus (1.75 BU/g), Lactiplantibacillus plantarum (0.5 BU/g), Saccharomyces boulardii (1.5 BU/g), as well as of the strains alone was assessed using Trypan Blue (Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA). The lyophilized probiotics were reconstituted in FBS- and antibiotics-free DMEM (PAN Biotech, Aidenbach, Germany) and incubated in 5% CO₂ at 37 °C for 48 h. Then, 10 μL of the probiotic solution was mixed with 10 μL Trypan Blue both at the beginning of the incubation (0 h) and, after 48 h, placed on a microscope slide and photographed at 40× and 100× using a light microscope (Leica DM2000, Leica Microsystems GmbH, Wetzlar, Germany).