Ph 123 microprocessor ph meter

To allow spheroid formation, 3.0 × 10⁵ colonic MSCs or colon cancer MSCs were transferred in 0,5 ml serum-free medium [55 (link)] in polypropylene 15 ml conical tubes (Falcon, BD) and incubated with or without CRC exosomes in 1:1000 (exosome mg/cell number) ratio until 72 hours at 37°C and 5% CO₂ in continuous rotation. Then cell spheroids were transferred in chamber slides (Falcon, BD), fixed in 3% paraformaldehyde, washed and analysed by a Nikon Eclipse T100 inverted microscope (Nikon Instruments Inc.) or by CLSM. The volume of the cMSCs spheroid structures were calculated using the following formula: (4π/3)xa²c, where a is the equatorial radius of the spheroid, c is the distance from center to pole along the symmetry axis. The pH of cMSCs spheroids supernatant was estimated by the use of a pH 123 Microprocessor pH Meter (Hanna Instruments, Italy).

Lansoprazole was purchased by Sigma-Aldrich (Milan, Italy) and was dissolved at 20 mM in DMSO in absence of direct light and reconstituted immediately prior its use. Efavirenz was purified from commercially available Sustiva (Bristol-Myers Squibb) as described [3 (link)], dissolved in DMSO and added to the culture medium in the indicated concentrations and times. RPMI-1640 cell culture medium (BE12-702F), antibiotics (DE17-603E), phosphate buffer saline (PBS) (BE17-512F), trypsin/EDTA (BE17-171E) and fetal bovine serum (DE14-701F) were obtained from Lonza (Milan, Italy). Trypan blue was bought from Alexis Biochemicals (Florence, Italy) and Annexin V-FITC Apoptosis detection kit from Enzo Life Sciences (Lausen, Switzerland). 4-Nitrophenyl phosphate disodium salt hexahydrate tablets for proliferation assay was from Sigma-Aldrich.
The pH of all cell culture supernatants were estimated by the use of a pH 123 Microprocessor pH Meter (Hanna Instruments, Italy).