Fiveeasyplus fep20 ph meter

Cryo-TEM
experiments were conducted using a CryoTitan system (Thermo Fisher
Scientific) equipped with a field emission gun and autoloader, operating
at a 300 kV acceleration voltage in low-dose bright-field TEM mode.
To prepare samples for cryo-TEM, grids (Lacey carbon coated, R2/2,
Cu, 200 mesh, EM sciences) were glow-discharged in a Cressington 208
carbon coater for 40 s. Subsequently, 4 μL of the samples was
pipetted onto the grid and blotting was performed in a Vitrobot MARK
III at room temperature and 100% humidity. The grid was blotted for
3 s (offset −3) and then directly plunged and frozen in liquid
ethane. Cryo-TEM images were acquired in zero-loss energy filtering
mode (Gatan GIF 2002, 20 eV energy slit) using a CCD camera (Gatan
model 794). Dynamic light scattering (DLS) experiments were carried
out on a Malvern Z90 Zetasizer instrument equipped with a 633 nm He–Ne
laser. An avalanche photodiode detector was utilized to characterize
the hydrodynamic size of the particles, and scattering light at a
173° angle was detected for size and distribution analysis. The
pH change of the solution was monitored using a Mettler Toledo FiveEasy
Plus FEP20 pH Meter. Confocal laser scanning microscopy (CLSM) imaging
of cell samples was performed with a Leica TCS SP8X.

The human colon adenocarcinoma cell lines, LS180 and Caco-2, were purchased from European Collection of Cell Cultures (ECACC) collections (KAC, Hyogo, Japan). LS180 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) containing 1,500 mg/L glucose (FUJIFILM Wako Pure Chemical, Osaka, Japan) and Caco-2 cells were cultured in DMEM with 4,500 mg/L glucose. In both the cases, the medium was supplemented with heat-inactivated 10% fetal bovine serum (FBS) (Cosmo Bio, Tokyo, Japan). The cultures were maintained at 37°C in a humidified atmosphere with 5% CO₂.
The acidic cell culture medium (pH 6.5) was obtained by the addition of 35–37% HCl solution to DMEM containing 1,500 mg/L glucose supplemented with heat-inactivated 10% FBS by measuring pH with a FiveEasy Plus FEP20 pH Meter (METTLER TOLEDO, Tokyo, Japan). LS180 cells were grown for a sufficient time in normal medium and were subsequently maintained in the acidic cell culture medium (pH 6.5) for a few days as described previously with some modifications [26 (link)–28 (link)].

Overnight 5-mL cultures of the WT and ypfP mutant were started from single colonies and grown at 37°C with shaking. Cells were pelleted, washed in PBS, and normalized to an OD₆₀₀ of 1.0. Cultures (100 mL) were inoculated to a starting OD₆₀₀ of 0.01 and grown anaerobically at 37°C in a Coy anaerobic chamber. Ten-milliliter volumes of the WT and ypfP cultures were collected at 0-, 2-, 4-, 6-, 8-, 12- and 24-h time points. A 1-mL volume of each time point sample was used to measure OD₆₀₀ with a Genesys 140 Visible spectrophotometer (Thermo Fisher Scientific). The remaining 9 mL of sample was pelleted, and the supernatant was filtered. The pH of the sterile supernatant was measured using a FiveEasy Plus FEP20 pH meter (Mettler Toledo).