Live dead stain kit

Melanoma cells were harvested at the indicated time points after transfection and once washed with PBS, followed by staining with live/dead stain kit (Invitrogen) and incubation at room temperature in the absence of light for 30 min. After washing, cells were split up for intracellular and surface staining. For surface staining, cells were incubated with the following antibodies: anti-AXL-SB436 (clone DS7HAXL, Invitrogen), anti-HLA-ABC-APC (clone W6/32, Invitrogen), anti-CD271(NGFR)-PE (clone ME20.4, BioLegend) and fixed with 4% paraformaldehyde. For intracellular staining, cells were fixed and permeabilized using Fixation/Permeabilization Kit (eBioscience) and stained with the following antibodies: anti-Melan-A-FITC (clone A103, Santa Cruz) and anti-CD271(NGFR)-APC/Fire750 (clone ME20.4, BioLegend). Fixed samples were measured using the Gallios flow cytometer (Beckman Coulter). Autofluorescence was determined by unstained samples, and Kaluza software (Beckman Coulter) was used for data analysis. Fold change in relative mean fluorescence intensity (MFI) was calculated by normalizing the MFI of treated cells to the corresponding controls.

One milliliter of bacterial suspension at a concentration of 1 × 10⁶ CFU/mL was added to a 24-well plate and co-cultured with metal disks for 24 h. The bacterial suspension was collected and fluorescently stained using the Live/Dead stain kit (Invitrogen, Eugene, OR), which contains two staining solutions, EthD-1 and AM, in which EthD-1 causes red fluorescence in dead bacteria, while AM causes green fluorescence in live bacteria, thereby differentiating between live and dead bacteria to assess inhibition performance. After staining for 15 min at room temperature under low light, the bacteria were observed under a confocal laser scanning microscope (CLSM, Leica TCS, SP2, Germany) for live and dead staining to assess the inhibition performance of the suspension.