Glass bottom culture dishes

KB Cells maintained in folate-depleted RPMI supplemented with 10% FBS and 1% penicillin/streptomycin were seeded (15,000 ​cells/well) onto glass bottom culture dishes (MatTek) and allowed to attach overnight. The next day, medium was removed, and cells were rinsed with PBS (200 ​μL ​× ​3) followed by the incubation of cells with 1 ​μM of commercial folic acid in 200 ​μL PBS (taken from a stock of 5 ​mM in DMSO) for 15 ​min at 37C, 5% CO_2. Next, the folic acid solution was pipetted out and the resulting KB cells were incubated with B-probe 1 (prepared according to the procedure described in earlier section) for 20 ​min. Finally, the excess/unbound bacteria were gently washed off with PBS containing Ca²⁺ ​and Mg²⁺ (3 ​× ​200 ​μL) and the cells were imaged using a fluorescence microscope with 60 ​× ​objective lens.

Cells (15,000 ​cells/well) were seeded onto glass bottom culture dishes (MatTek) and allowed to adhere overnight. MDA-MB-435 ​cells were seeded in MEM medium supplemented with 5% charcoal dextran-stripped calf serum, 1% non-essential amino acids, 1% l-glutamine, and 1% penicillin/streptomycin. KB cells were plated in folate-depleted RPMI supplemented with 10% FBS and 1% penicillin/streptomycin while LNCaP were maintained in RPMI, 10% FBS, 1% l-glutamine, and 1% penicillin/streptomycin. Following day, medium was removed, rinsed twice with PBS and then the cells were incubated with B-probes 1–4 in a CO₂ incubator (5% CO₂) at 37C for 20 ​min. Finally, the excess/unbound bacteria were gently washed off with PBS containing Ca²⁺ ​and Mg²⁺ (3 ​× ​200 ​μL) and the cells were imaged using a fluorescence microscope with 60 ​× ​objective lens. Negative control experiments were performed similarly using bacteria decorated with a duplexes generated from tri-NTA-ODN₁ and DNA strands (lacking the corresponding CSP binders) namely, TAMRA-ODN₂, Cy5-ODN₂ or FAM-ODN₂.

KB, MDA-MB-435 ​cells, and LNCaP cells were maintained in their respective media as mentioned in the previous section. Cells (15,000 ​cells/well) were seeded onto glass bottom culture dishes (MatTek) and allowed to adhere overnight. The medium was removed from the cells and rinsed twice with PBS. On the other hand, B-probes 1–3 (prepared as described in the earlier section) were mixed (3 ​× ​200 ​μL). A mixture of this bacterial sample (200 ​μL) was incubated separately with each cell line at 37 ​°C under 5% CO₂ condition. After 20 ​min, the unbound bacteria were gently washed off with PBS containing Ca⁺² and Mg⁺² (3 ​× ​200 ​μL) and the cells were imaged using a fluorescence microscope and a 60 ​× ​objective lens.

Sensory neurons of trigeminal ganglia (TGs) were obtained from 8 to 12 week-old Trpm3^+/+ and Trpm3^−/− mice, as described before [32 (link),37 (link)]. Briefly, mice were euthanized by CO₂, TGs were isolated and digested with collagenase (2 mg/ml) and dispase (2,5 mg/ml) (both from Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA). Suspension of sensory neurons was seeded on laminin (100 μg/ml) and poly-L-ornithine HBr (500 μg/ml) (both from Sigma Aldrich) coated glass bottom culture dishes (MatTek, Ashland, MA, USA) and cultured in Neurobasal medium supplemented with 2% B-27 supplement, 2 mM L-glutamine, 100 μg/ml penicillin/streptomycin, 2 ng/ml glial cell line-derived neurotrophic factor (GNDF) (all from Invitrogen/Thermo Fisher Scientific) and 10 ng/ml NT-4 (PeproTech, London, UK) at 37 °C in 5% CO₂ containing humidified atmosphere. Neurons were used for experiments within 24 to 36 hrs following isolation.