Astrocyte cell lysate preparation

Aliquots of astrocyte lysates, each containing about 10⁶ cells/ml, were centrifuged at 800 x g for 10min at RT in 1.5ml Eppendorf tubes. Supernatants were removed, and cell pellets were suspended in 100μl of lysis buffer containing 137mM NaCl, 20mM TRIS-HCl (pH 7.5), 0.5% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, 1mM phenylmethylsulphonyl fluoride, 10% glycerol, 2mM EDTA, 0.15U/ml aprotinin, 25mM β-glycerophosphate and 1mM sodium orthovanadate. Cell suspensions were then lysed by shock freezing in liquid nitrogen and kept at -70°C.