Methyl 3h thymidine 50 ci mmol

[³H]-thymidine incorporation was measured to determine the effect on rat mesnagial cell proliferation. Quiescent cells were treated with 25 mM glucose and Oryeongsan, respectively, and 1 μCi of [³H]-thymidine was added (methyl-[³H] thymidine 50 Ci/mmol; Amersham, Oakville, Ontario, Canada). After incubation for 24 h, cells were washed once with 2 ml of ice-cold PBS for 10 minutes, extracted three times with 2 ml of cold 10% TCA for 5 minutes each time, and solubilized for at least 30 minutes at room temperature in 0.2 ml 0.3 N NaOH, 1% SDS. After neutralizing with 0.2 ml 0.3 N HCI, [³H]-thymidine activity was measured in a liquid scintillation counter (Beckman LS 7500, Fullerton, CA). Each experiment was performed in triplicate or quadruplicate.

[³H]-thymidine incorporation was measured to determine the effect on human mesangial cell proliferation. The mesangial cells were plated at 2.5 × 10⁴ cells/well in 24-well cell culture plates for 24 h, and the medium was replaced with serum-free medium containing 0.1% bovine serum albumin and the incubation continued for another 24 h. Quiescent cells were treated with 10 μM angiotensin II and DS-EA, respectively, and 1 µCi of [³H]-thymidine was added (methyl-[³H] thymidine 50 Ci/mmol; Amersham, Oakville, ON, Canada). After incubation for 24 h, cells were extracted three times with cold 10% TCA for 5 min each time and solubilized for at least 30 min in 0.3 N NaOH. After neutralizing, [³H]-thymidine activity was measured in a liquid scintillation counter (Beckman LS 7500, Fullerton, CA, USA). Each experiment was performed in triplicate or quadruplicate. Mesangial cell index was calculated for each E-plate well by RTCA Software 1.2 (Roche Diagnosis, Meylan, France). The graphs are real-time generated outputs from the iCELLigence system.