Primaria culture dish

COS-7 cells were maintained in Dulbecco's modified Eagle medium (DMEM), containing 10% heat-inactivated fetal calf serum (FCS). PC12 cells were grown in DMEM containing 10% heat-inactivated horse serum (HS) and 5% heat-inactivated FCS using a PRIMARIA culture dish (BD Falcon).
Mouse hippocampal neurons were prepared from P0 ICR mice, as described previously (Bito et al., 1996 (link)). Dissociated cells were plated onto 12-mm Matrigel-coated coverslips, transfected with each expression vector using Lipofectamine 2000 (Invitrogen) at 7 days in vitro, and fixed 2–3 days later for immunostaining. Neurons were treated with either 60 μM 2-bromopalmitate (Sigma) overnight or 10 mM methyl-β-cyclodextrin (Sigma) for 20 min before fixation (Figures 1C, D).

Determination of potential increase in intracellular K⁺ concentration from its control value by an increase in extracellular K⁺ concentration or by dobutamide was determined as by Xu et al. [17 (link)], using the K⁺-sensing fluorescent drug PBFI-AM. At the start of the experiment astrocytes cultured on coverslips coated with polylysine and placed within a Falcon Primaria culture dish were loaded with 10 μM PBFI-AM with 0.2% Pluronic F-127 in phosphate-buffered saline (concentrations in mM: Na⁺ 141, K⁺ 5.4, Cl⁻ 146, PO₄³⁻ 0.44, HCO₃⁻ 4.0, Ca²⁺ 1.3, Mg²⁺ 1.3, SO₄²⁻, and glucose 10) for 45 min at 37°C. After 2 times of wash with similar saline, the coverslip was superfused with saline solution with or without Ca²⁺ for 2 min, after which KCl was added. Subsequently the fluorescence intensity was recorded in each of ~20 individual cells for 7 min with 20 sec interval at excitation wavelengths of 340 (F340) and 380 (F380) nm. The ratio F340/F380 is an arbitrary measurement of the intracellular K⁺ concentration, and its potential changes during the measurement are an indication of K⁺ uptake rates. No calibration was made to obtain absolute values.