Fv1000d laser scanning confocal microscope

Cells were grown on sterilized 12-holes microslide glass (Matsunami, Osaka, Japan) at 1.5 × 10³ cells/hole overnight. Cells were washed twice with calcium free PBS (PBS-) and probed with primary antibodies against heat shock protein 60 (HSP60) (D6F1, Cell Signaling Technology, Beverly, MA, USA) and GLUT1 (CSB-PA002728, Cusabio Biotech, College Park, MD, USA) at 4 °C for 30 min. Cells were subsequently stained with goat anti-rabbit IgG Hilyte Fluor 647 (AnaSpec, Fremont, CA, USA). CD44 was stained by PE conjugated antibody (IM7, BioLegend, San Diego, CA, USA). Additionally, 4′,6-diamidino-2-phenylindole (DAPI) (Wako) was used for staining the nucleus. Expression and localization of proteins were examined using an FV1000D laser scanning confocal microscope (Olympus, Tokyo, Japan).

Exponentially growing HeLa S3 cells were seeded on an 8-well glass chamber slide coated with collagen (Lab-Tek™, Nunc) at 1.2 × 10⁴ cells per 0.25 mL. The cells were incubated for 24 h at 37 °C prior to the addition of samples. Cells that had been treated with samples for 6 h at 37 °C were washed with PBS and fixed with MeOH (250 μL) for 30 min at −20 °C. After being washed with PBS, the cells were blocked with 0.5% BSA in PBS for 1 h at room temperature. The cells were then incubated in anti-α-tubulin monoclonal antibody DM1A (cat. no. sc-32293, Santa Cruz Biotechnology) at 0.8 μg/mL, diluted in the blocking buffer, for 1 h at room temperature. The cells were washed with 0.1% BSA/PBS and incubated in Alexa Fluor^® 488 anti-mouse IgG (Invitrogen) at 2 μg/mL, diluted in 0.1% BSA/PBS. The mixture was left to stand for 1 h at room temperature. After being washed four times with PBS, 4′,6-diamidino-2-phenylindole (DAPI, DOJINDO) at 0.5 μg/mL in PBS was added, and the mixture was left to stand for 1 h. The cells were again washed with PBS, the polystyrene chambers were removed, and slides were mounted with SlowFade^® Gold antifade reagent (Invitrogen). Fluorescence and bright-field images of fixed cells were captured using an Olympus FV1000-D laser scanning confocal microscope.

Ovarian cancer cells were transfected with precursor miRNA (pre-hsa-miR-199a-3p, #PM11779) or inhibitor miRNA (anti-hsa-miR-199a-3p, #AM11779) at a concentration of 166 nM. Pre-miR miRNA Precursor Negative Control #1 (#AM17110) was used as a control, and FAM-labeled Pre-miR Negative Control #1 (#AM17121) was used to confirm the transfection efficacy. All oligonucleotides were purchased from Life Techonologies. Oligonucleotide transfection was performed using Lipofectamine 2000 in accordance with the manufacturer's instructions. Twenty-four hours after transfection, cells were collected for subsequent analysis. Transfection efficacy was confirmed by detecting FAM-labeled cells using a FV1000-D Laser Scanning Confocal Microscope (Olympus, Tokyo, Japan).

J774.1 cells on culture slides were infected with bacteria at a multiplicity of infection (MOI) of 10 for 30 min and then incubated with 10 μM of inhibitors for 90 min. Cells fixed with methanol for 4 min at -20°C were stained with Alexa Fluor 594 conjugated antibody against CD107a, known as lysosomal-associated membrane protein-1 (LAMP1) (Cosmo Bio), for 45 min at room temperature (Chen et al., 1985 (link)). Slides were observed using an FV1000-D laser scanning confocal microscope (Olympus, Tokyo, Japan). One hundred randomized events were analyzed for each drug, which were repeated three times and statistically analyzed. The rate of lysosomal transfer (%) was calculated as follows: lysosomal transfer (%) = 100 × (number of bacteria overlapping LAMP-1 / total number of analyzed bacteria.)