Microcon ym 10 centrifugal filters

The GCPs (3 × 10⁶ cells) were suspended in 0.4 M mannitol, 10 mM KCl, 0.5 mM CaCl₂, 0.5 mM MgCl₂ and 5 mM MES-Tris buffer (pH 6.0) and illuminated with white light (80 μmol m^-2 s^-1 for 1 h) as described elsewhere [22 (link)] before isolation of the ions. The GCPs were ground into a fine powder, suspended in distilled water and boiled for 5 min. The suspensions were applied to Microcon YM-10 centrifugal filters (Millipore), and the organic/inorganic ions were measured in the filtrate.
The measurement of organic and inorganic ions was performed on an HPLC (Prominence, Shimadzu). The chromatographic system consisted of a Shimadzu Model system controller (SLC-10AVP), conductivity detector (CDD-10ADVP), and column oven (CTO-20A). K^- and Na⁺ were separated using a LC-10ADVP pump, a column Shim-pack IC-C4 (150 mm × 4.6 mm) and a guard column Shim-pack IC-GC4 (10 mm × 4.6 mm) with a mobile phase (3.2 mM Bis-Tris, 8 mM p-hydroxybenzoic acid, 50 mM Boric acid) and a flow rate of 1.0 ml min^-1 at 40°C. Cl^- and Malate^2- were separated with a LC-20AD pump, a column Shim-pack IC-A3 (150 mm × 4.6 mm) and guard column Shim-pack IC-GA3 (10 mm × 4.6 mm) with a mobile phase (3/4 dilution of 3.2 mM Bis-Tris, 8 mM p-hydroxybenzoic acid, 50 mM boric acid) and a flow rate of 1.2 ml min^-1 at 40°C. Statistical analyses were performed using the Student's t test.

L-α-Phosphatidylcholine from soybean (LEC), cholesterol (CHOL) and 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC) were purchased from Sigma-Aldrich Ltd. (Budapest, Hungary). All the excipients used in our experiments (hydroxyethyl-cellulose (HEC), phenyl salicylate, caffeine, sodium dihydrogen phosphate, sodium monohydrogen phosphate, polyvinyl alcohol (PVA)) were from Hungaropharma Ltd. (Budapest, Hungary). Deionized ultrafiltered water was produced by Milli-Q system (Millipore Inc., Budapest, Hungary). Polycarbonate filters with a pore size of 200 nm applied for extrusion were purchased from Schleicher & Schuell GmbH. (Dassel, Germany). Microcon YM-10 centrifugal filters with a cut-off value of 10 kDa were purchased from Millipore Inc. (Budapest, Hungary).