Kit 23200

The uptake of [3H]-glutamic acid was used to determine change in glutamate uptake experienced by astrocytes on soft and stiff surface. The media was removed and replaced by serum free high glucose DMEM containing 50 μM glutamate and 18.5 kBq of [3H]-glutamic acid [Perkin Elmer] which was allowed to incubate at 37 °C of 15 min. Uptake was terminated by removal of working solution and cells washed twice with ice-cold PBS lysed in 10 mM NaOH containing 0.1% Triton X-100. 300 ml of lysate was added to liquid scintillation cocktail [Fisher Scientific] and quantified by counting. The protein content was assayed using Bradford assay [Thermo Scientific Kit 23200]. Results were reported as CPM per mg protein.

Whole cell lysates were collected using a RIPA buffer (PBS, pH 7.4, 1% CA 630 IGEPAL, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate, protease inhibitor cocktail and phenylmethylsufonyl fluoride) [Sigma Aldrich]. Proteins were quantified using coomassie blue [Thermo Scientific Kit 23200]. 10–50 μg of total protein was separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to Immobilon FL membrane [Millipore] using transfer buffer (25 mM Tris, 192 mM glycine, 10% methanol) and detected with primary antibodies (GAPDH [Millipore], GFAP [DAKO], EAAT1 [Abcam], EAAT2 [Abcam] and vimentin [GeneTex]) followed by Dylight 800 polyclonal secondary antibody [Thermo Scientific] and imaged with an Odyssey FC [LiCor].